Combined cancer treatment methods using antibodies to aminophospholipids

ABSTRACT

Disclosed are the surprising discoveries that aminophospholipids, such as phosphatidylserine and phosphatidylethanolamine, are stable and specific markers accessible on the luminal surface of tumor blood vessels, and that the administration of an anti-aminophospholipid antibody alone is sufficient to induce thrombosis, tumor necrosis and tumor regression in vivo. This invention therefore provides anti-aminophospholipid antibody-based methods and compositions for use in the specific destruction of tumor blood vessels and in the treatment of solid tumors. Although various antibody conjugates and combinations are thus provided, the use of naked, or unconjugated, anti-phosphatidylserine antibodies is a particularly important aspect of the invention, due to simplicity and effectiveness of the approach.

[0001] The present application claims priority to first provisionalapplication Ser. No. 60/092,672, filed Jul. 13, 1998, and sec ndprovisional application Ser. No. 60/110,608, filed Dec. 02, 1998, theentire text and fi res of which applications are incorporated herein byreference without disclaimer. The U.S. Government owns rights in thepresent invention pursuant to grant numbers IROICA 74951-0, and5ROICA54168-05 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of bloodvessels and tumor biology. More particularly, it embodies the surprisingfindings that aminophospholipids, such as phosphatidylserine andphosphatidylethanolamine, are specific and stable markers of tumor bloodvessels and that administration of anti-aminophospholipid antibodiesalone is sufficient to induce thrombosis and tumor regression. Theinvention thus provides safe and effective methods and compositions forthe specific targeting and destruction of tumor blood vessels and forthe treatment of solid tumors. The use of unconjugatedanti-phosphatidylserine antibodies is a particular advantage, althoughthe invention provides various effective compositions and combinationsthereof.

[0004] 2. Description of the Related Art

[0005] Tumor cell resistance to chemotherapeutic agents represents asignificant problem in clinical oncology. In fact, this is one of themain reasons why many of the most prevalent forms of human cancer stillresist effective chemotherapeutic intervention, despite certain advancesin the field of chemotherapy.

[0006] A significant problem to address in tumor treatment regimens isthe desire for a “total cell kill”. This means that the more effectivetreatment regimens come closer to a total cell kill of all so-called“clonogenic” malignant cells, i.e., cells that have the ability to growuncontrolled and replace any tumor mass that might be removed by thetherapy. Due to the goal of developing treatments that approach a totalcell kill, certain types of tumors have been more amenable to therapythan others. For example, the soft tissue tumors, e.g, lymphomas, andtumors of the blood and blood-forming organs, e.g., leukemias, havegenerally been more responsive to chemotherapeutic therapy than havesolid tumors, such as carcinomas.

[0007] One reason for the susceptibility of soft and blood-based tumorsto chemotherapy is the greater accessibility of lymphoma and leukemiccells to chemotherapeutic intervention. Simply put, it is much moredifficult for most chemotherapeutic agents to reach all of the cells ofa solid tumor mass than it is the soft tumors and blood-based tumors,and therefore much more difficult to achieve a total cell kill.Increasing the dose of chemotherapeutic agents most often results intoxic side effects, which generally limits the effectiveness ofconventional anti-tumor agents.

[0008] Another tumor treatment strategy is the use of an “immunotoxin”,in which an anti-tumor cell antibody is used to deliver a toxin to thetumor cells. However, in common with the chemotherapeutic approachesdescribed above, immunotoxin therapy also suffers from significantdrawbacks. For example, antigen-negative or antigen-deficient cells cansurvive and repopulate the tumor or lead to further metastases. Also, inthe treatment of solid tumors, the tumor mass is generally impermeableto molecules of the size of antibodies and immunotoxins. Both thephysical diffusion distances and the interstitial pressure within thetumor are significant limitations to this type of therapy.

[0009] A more recent strategy has been to target the vasculature ofsolid tumors. Targeting the blood vessels of the tumors, rather than thetumor cells themselves, has certain advantages in that it is not likelyto lead to the development of resistant tumor cells, and that thetargeted cells are readily accessible. Moreover, destruction of theblood vessels leads to an amplification of the anti-tumor effect, asmany tumor cells rely on a single vessel for their oxygen and nutrients(Denekamp, 1990). Effective vascular targeting strategies are describedin U.S. Pat. Nos. 5,855,866 and 5,___,___ (U.S. application Ser. No.08/350,212, Issue Fee paid), which particularly describe the targeteddelivery of anti-cellular agents and toxins to tumor vasculature.

[0010] Another effective version of the vascular targeting approach isto target a coagulation factor to tumor vasculature (Huang et al, 1997;U.S. Pat. Nos. 5.877,289, 5,___,___ and 5,___,___ (U.S. application Ser.Nos. 08/487,427 and 08/482,369; Issue Fees paid)). The use of antibodiesand other targeting agents to deliver coagulants to tumor vasculaturehas the further advantages of reduced immunogenicity and even lower riskof toxic side effects. As disclosed in U.S. Pat. No. 5,877,289, apreferred coagulation factor for use in such tumor-specific thrombogens,or “coaguligands”, is a truncated version of the humancoagulation-inducing protein, Tissue Factor (TF). TF is the majorinitiator of blood coagulation (Ruf et al., 1991; Edgington et al.,1991; Ruf and Edgington, 1994). Treatment of tumor-bearing mice withsuch coaguligands results in significant tumor necrosis and evencomplete tumor regression in many animals (Huang et al., 1997; U.S. Pat.Nos. 5,877,289, 5,___,___ and 5,___,___ (U.S. applications Ser. Nos.08/487,427 and 08/482,369; Issue Fees paid)).

[0011] Although the specific delivery of therapeutic agents, such asanti-cellular agents, toxins and coagulation factors, to tumor vesselsrepresents a significant advance in tumor treatment protocols, there isstill room for additional or even alternative vascular targetingtherapies. The identification of additional targets to allow specifictumor vessel destruction in vivo would naturally be of benefit inexpanding the number of targeting options. More particularly, as thepreviously described vascular targeting constructs and coaguligands aretwo-component systems, involving the targeting agent and the effectorportion, the development of a one component agent for tumor vasculaturedestruction would represent a major advance. Should the preparation ofthis type of agent prove possible, this would also likely speed theprogress of anti-vascular therapy to the clinic, given the simplicity ofthe new therapeutic agent.

SUMMARY OF THE INVENTION

[0012] The present invention addresses the needs of the prior art byproviding new, simplified therapeutic methods for specific tumordestruction. The invention is based, in part, on the finding thataminophospholipids, such as phosphatidylserine andphosphatidylethanolamine, are accessible and stably targetable markersof tumor vasculature. More particularly, the invention embodies theunexpected discovery that naked antibodies against aminophospholipidcomponents are capable of specifically inducing tumor blood vesseldestruction and tumor necrosis in vivo.

[0013] Certain preferred aspects of the invention were developed fromthe surprising discovery that antibodies against the aminophospholipid,phosphatidylserine (PS), specifically localize to the vasculature ofsolid tumors and, even more surprisingly, exert a tumor destructiveeffect in the absence of conjugation to effector molecules, such astoxins or coagulants. Single component therapeutics directed againstaminophospholipids thus represent a breakthrough in vascular targetingand provide safe and effective methods for the treatment of solidtumors.

[0014] An underlying surprising feature of the invention is thattranslocation of aminophospholipids, such as PS, to the surface of tumorvascular endothelial cells occurs, at least in a significant part,independently of cell damage and apoptopic or other cell-deathmechanisms. PS surface expression in the tumor environment is thereforenot a consequence of, or a trigger for, cell death and destruction, butoccurs on morphologically intact vascular endothelial cells. This meansthat PS expression is not transient, but rather is stable enough toprovide a target for therapeutic intervention.

[0015] The methods of the invention thus provide for killing, orspecifically killing, tumor vascular endothelial cells, and compriseadministering to an animal or patient having a vascularized tumor atleast one dose of a biologically effective amount of at least a firstpharmaceutical composition comprising a naked or unconjugated antibody,or antigen-binding region thereof, that binds to at least a firstaminophospholipid expressed on the luminal surface of tumor vascularendothelial cells. The “biologically effective amount” is an amount ofthe naked or unconjugated antibody effective to specifically kill atleast a portion, and preferably a significant portion, of the tumorvascular endothelial cells, as opposed to endothelial cells in normalvessels, upon binding to an aminophospholipid expressed on the luminalsurface of the tumor vascular endothelial cells. As such, it is an“endothelial cell killing amount” or a “tumor vascular endothelial cellkilling amount” of a naked or unconjugated anti-aminophospholipidantibody or antigeni-binding region thereof.

[0016] As used throughout the entire application, the terms “a” and “an”are used in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components, except ininstances wherein an upper limit is thereafter specifically stated.Therefore “an anti-aminophospholipid antibody” means “at least a firstanti-aminophospholipid antibody”.

[0017] The operable limits and parameters of combinations, as with theamounts of any single agent, will be known to those of ordinary skill inthe art in light of the present disclosure.

[0018] The “a” and “an” terms are also used to mean “at least one”, “atleast a first”, “one or more” or “a plurality” of steps in the recitedmethods, except where specifically stated. This is particularly relevantto the administration steps in the treatment methods. Thus, not only maydifferent doses be employed with the present invention, but differentnumbers of doses, e.g., injections, may be used, up to and includingmultiple injections.

[0019] An “aminophospholipid”, as used herein, means a phospholipid thatincludes within its structure at least a first primary amino group.Preferably, the term “aminophospholipid” is used to refer to a primaryamino group-containing phospholipid that occurs naturally in mammaliancell membranes. However, this is not a limitation on the meaning of theterm “aminophospholipid”, as this term also extends to non-naturallyoccurring or synthetic aminophospholipids that nonetheless have uses inthe invention, e.g., as an immunogen in the generation ofanti-aminophospholipid antibodies (“cross-reactive antibodies”) that dobind to aminophospholipids of mammalian plasma membranes. Theaminophospholipids of U.S. Pat. No. 5,767,298, incorporated herein byreference, are appropriate examples.

[0020] The prominent aminophospholipids found in mammalian biologicalsystems are the negatively-charged phosphatidylserine (“PS”) and theneutral or zwitterionic phosphatidylethanolamine (“PE”), which aretherefore preferred aminophospholipids for targeting by the presentinvention. However, the invention is by no means limited to thetargeting of phosphatidylserines and phosphatidy lethaiiolani nes, andany other aminophospholipid target may be employed (White et til.* 1978;incorporated herein by reference) so long as it is expressed, accessibleor complexed on the luminal surface of tumor vascular endothelial cells.

[0021] All aminophospholipid-, phosphatidylserine- andphosphatidylethanolamine-based components are encompassed as targets ofthe invention irrespective of the type of fatty acid chains involved,including those with short, intermediate or long chain fatty acids, andthose with saturated, unsaturated and polyunsaturated fatty acids.Preferred compositions for raising antibodies for use in the presentinvention may be aminophospholipids with fatty acids of C18, with C18:1being more preferred (Levy et al., 1990; incorporated herein byreference). To the extent that they are accessible on tumor vascularendothelial cells, aminophospholipid degradation products having onlyone fatty acid (lyso derivatives), rather than two, may also be targeted(Qamar et al., 1990; incorporated herein by reference).

[0022] Another group of potential aminophospholipid targets include, forexample, phosphatidal derivatives (plasmalogens), such asphosphatidalserine and phosphatidalethanolamine (having an ether linkagegiving an alkenyl group, rather than an ester linkage giving an acylgroup). Indeed, the targets for therapeutic intervention by the presentinvention include any substantially lipid-based component that comprisesa nitrogenous base and that is present, expressed, translocated,presented or otherwise complexed in a targetable form on the luminalsurface of tumor vascular endothelial cells, not excludingphosphatidylcholine (“PC”). Lipids not containing glycerol may also formappropriate targets, such as the sphingolipids based upon sphingosineand derivatives.

[0023] The biological basis for including a range of lipids in the groupof targetable components lies, in part, with the observed biologicalphenomena of lipids and proteins combining in membranous environments toform unique lipid-protein complexes. Such lipid-protein complexes extendto antigenic and immunogenic forms of lipids such as phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine with, e.g., proteinssuch as P₂-glycoprotein I, prothrombin, kininogens and prekallikrein.Therefore, as proteins and polypeptides can have one or more freeprimary amino groups, it is contemplated that a range of effective“aminophospholipid targets” may be formed in vivo from lipid componentsthat are not aminophospholipids in the strictest sense. Nonetheless, allsuch targetable complexes that comprise lipids and primary amino groupsconstitute an “aminophospholipid” within the scope of the presentinvention.

[0024] The inventive methods also act to arrest blood flow, orspecifically arrest blood flow, in tumor vasculature. This is achievedby administering to an animal or patient having a vascularized tumor atleast one dose of at least a first pharmaceutical composition comprisinga coagulation-inducing amount, or a vessel-occluding amount, of at leasta first naked or unconjugated antibody, or antigen-binding regionthereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, translocated to theluminal surface of tumor vasculature.

[0025] A “coagulation-inducing amount” or “vessel-occluding amount” isan amount of the naked or unconjugated antibody effective tospecifically induce or promote coagulation in, and hence occlude, atleast a portion, and preferably a significant portion, of tumor orintratumoral blood vessels, as opposed to normal blood vessels, uponbinding to an aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, translocated to the luminal surface of tumorblood vessels. The “vessel-occluding amount” is therefore a functionallyeffective amount, and is not a physical mass of antibody sufficient tospan the breadth of a vessel.

[0026] Methods for destroying, or specifically destroying, tumorvasculature are provided that comprise administering to an animal orpatient having a vascularized tumor one or more doses of at least afirst pharmaceutical composition comprising a tumor-destructive amountof at least a first naked or unconjugated antibody, or antigen-bindingregion thereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, presented on the luminalsurface of tumor vasculature. The “tumor-destructive amount” is anamount of the naked or unconjugated antibody effective to specificallydestroy or occlude at least a portion, and preferably a significantportion, of tumor blood vessels, as opposed to normal blood vessels,upon binding to an amrinophosplholipid, preferably phosphatidylserinicor phosphatidylethanolamine, presented on the luminal surface of thevascular endothelial cells of the tumor blood vessels.

[0027] The invention further encompasses methods for treating cancer andsolid tumors, comprising administering to an animal or patient having avascularized tumor a tumor necrosis-inducing amount or amounts of atleast a first pharmaceutical composition comprising at least a firstnaked or unconjugated antibody, or antigen-binding fragment thereof,that binds to an aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, on the luminal surface of blood vessels of thevascularized tumor. The “tumor necrosis-inducing amount” is an amount ofthe naked or unconjugated antibody effective to specifically inducehemorrhagic necrosis in at least a portion, and preferably a significantportion, of the tumor upon binding to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, complexed at the luminalsurface of the vascular endothelial cells of the tumor blood vessels,while exerting little adverse side effects on normal, healthy tissues.

[0028] The methods of the invention may thus be summarized as methodsfor treating an animal or patient having a vascularized tumor,comprising administering to the animal or patient at least a first doseof a therapeutically effective amount of at least a first pharmaceuticalcomposition comprising at least a first naked or unconjugated antibody,or antigen-binding fragment thereof, that binds to an aminophospholipid(preferably phosphatidylserine or phosphatidylethanolamine) present,expressed, translocated, presented or complexed at the luminal surfaceof blood transporting vessels of the vascularized tumor.

[0029] The essence of the invention may also be defined as a compositioncomprising at least a first naked or unconjugated anti-aminophospholipidantibody, preferably an anti-phosphatidylserine oranti-phosphatidylethanolamine antibody, or antigen-binding fragmentthereof, for use in the preparation of a medicament for use in tumorvasculature destruction and for human tumor treatment. This can also bedefined as a composition comprising at least a first naked orunconjugated anti-aminophospholipid antibody, preferably an anti-PS oranti-PE antibody, or antigen-binding fragment thereof, for use in thepreparation of a medicament for use in binding to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof blood transporting vessels of a vascularized tumor and for use ininducing tumor vasculature destruction and for human tumor treatment.

[0030] In the methods, medicaments and uses of the present invention,one of the advantages lies in the fact that the provision of a simplenaked or unconjugated anti-aminophospholipid antibody composition,preferably anti-phosphatidylserine or anti-phosphatidylethanolamine,into the systemic circulation of an animal or patient results in thepreferential or specific destruction of the tumor vasculature and theinduction of tumor necrosis. The invention therefore solves the problemof the complex preparative methods of the multicomponent anti-vascularagents of the prior art.

[0031] The terms “naked” and “unconjugated” antibody, as used herein,are intended to refer to an antibody that is not conjugated, operativelylinked or otherwise physically or finctionally associated with aneffector moiety, such as a cytotoxic or coagulative agent. It will beunderstood that the terms “naked” and “unconjugated” antibody do notexclude antibody constructs that have been stabilized, multimerized,humanized or in any other way manipulated, other than by the attachmentof an effector moiety.

[0032] Accordingly, all post-translationally modified naked andunconjugated antibodies are included herewith, including where themodifications are made in the natural antibody-producing cellenvironment, by a recombinant antibody-producing cell, and areintroduced by the hand of man after initial antibody preparation. Ofcourse, the term “naked” antibody does not exclude the ability of theantibody to form functional associations with effector cells and/ormolecules after administration to the body, as some such interactionsare necessary in order to exert a biological effect. The lack ofassociated effector group is therefore applied in definition to thenaked antibody in vitro, not in vivo.

[0033] In the context of the present invention, the term “a vascularizedtumor” most preferably means a vascularized, malignant tumor, solidtumor or “cancer”. The invention is particularly advantageous intreating vascularized tumors of at least about intermediate size, and intreating large vascularized tumors—although this is by no means alimitation on the invention. The invention may therefore be used in thetreatment of any tumor that exhibits aminophospholipid-positive bloodvessels, preferably phosphatidylserine- and/orphosphatidylethanolamine-positive blood vessels.

[0034] In preferred embodiments, the tumors to be treated by the presentinvention will exhibit a killing effective number ofaminophospholipid-positive blood vessels. “A killing effective number ofaminophospholipid-positive blood vessels”, as used herein, means that atleast about 3% of the total number of blood vessels within the tumorwill be positive for aminophospholipid expression, preferablyphosphatidylserine and/or phosphatidylethanolamine expression.Preferably, at least about 5%, at least about 8%, or at least about 10%or so, of the total number of blood vessels within the tumor will bepositive for aminophospholipid expression. Given theaminophospholipid-negative, particularly PS-negative, nature of theblood vessels within normal tissues, the tumor vessels will act as sinkfor the administered antibodies. Furthermore, as destruction of only aminimum number of tumor vessels can cause widespread thrombosis,necrosis and an avalanche of tumor cell death, antibody localization toall, or even a majority, of the tumor vessels is not necessary foreffective therapeutic intervention.

[0035] Nonetheless, in more preferred embodiments, tumors to be treatedby this invention will exhibit a significant number ofaminophospholipid-positive blood vessels. “A significant number ofaminophospholipid-positive blood vessels”, as used herein, means that atleast about 10-12% of the total number of blood vessels within the tumorwill be positive for aminophospholipid expression, preferablyphosphatidylserine and/or phosphatidylethanolamine expression. Even morepreferably, the % of aminophospholipid-expressing tumor vessels will beat least about 15%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,or at least about 80% or so of the total number of blood vessels withinthe tumor, up to and including even at least about 90% or 95% of thevessels.

[0036] The “therapeutically effective amounts” for use in the inventionare amounts of naked or unconjugated anti-aminophospholipid antibodies,preferably anti-PS or anti-PE antibodies, effective to specifically killat least a portion of tumor vascular endothelial cells; to specificallypromote coagulation in at least a portion of tumor blood vessels; tospecifically occlude or destroy at least a portion of blood transportingvessels of the tumor; to specifically induce necrosis in at least aportion of a tumor; and/or to induce tumor regression or remission uponadministration to selected animals or patients. Such effects areachieved while exhibiting little or no binding to, or little or nokilling of, vascular endothelial cells in normal, healthy tissues;little or no coagulation in, occlusion or destruction of blood vesselsin healthy, normal tissues; and exerting negligible or manageableadverse side effects on normal, healthy tissues of the animal orpatient.

[0037] The terms “preferentially” and “specifically”, as used herein inthe context of promoting coagulation in, or destroying, tumorvasculature, and/or in the context of causing tumor necrosis, thus meanthat anti-aminophospholipid antibodies function to achieve coagulation,destruction and/or tumor necrosis that is substantially confined to thetumor vasculature and tumor site, and does not substantially extend tocausing coagulation, destruction and/or tissue necrosis in normal,healthy tissues of the animal or subject. The structure and function ofhealthy cells and tissues is therefore maintained substantiallyunimpaired by the practice of the invention.

[0038] Therapeutic benefits may be realized by the administration of atleast two, three or more naked or unconjugated anti-aminophospholipidantibodies; bispecific antibodies; chimeric antibodies; and/or dimeric,trimeric or multimeric antibodies. The anti-aminophospholipid antibodiesmay also be combined with other therapies to provide combinedtherapeutically effective amounts, as disclosed herein.

[0039] Although understanding the mechanism of action is not necessaryto the practice of the present anti-aminophospholipid antibody treatmentinvention, the methods may operate to induce cell-mediated cytotoxicity,complement-mediated lysis and/or apoptosis. The cytotoxic methods mayalso be based upon antibody-induced cell signaling (direct signaling),or mimicking or altering signal transduction pathways (indirectsignaling). The ability of the anti-amilophospholipid antibodies tolocalize and bind to a component of the membrane itself may also berelevant, as opposed to previous therapies that are generally directedto binding to a protein component or complex, which may be stericallydistinct or distant from the membrane surface itself.

[0040] The treatment methods thus include administering to an animal orpatient having a vascularized tumor at least a first pharmaceuticalcomposition comprising an amount of at least a first antibody constructeffective to induce, or specifically induce, cell-mediated cytotoxicityof at least a portion of the tumor vascular endothelial cells. Herein,the first antibody construct is a naked or unconjugated antibody, oreffective fragment thereof, that binds to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof tumor vascular endothelial cells and that induces cell-mediatedcytotoxicity of at least a portion of the tumor vascular endothelialcells, as opposed to endothelial cells in normal vessels. As usedherein, “cell-mediated cytotoxicity or destruction” includes ADCC(antibody-dependent, cell-mediated cytotoxicity) and NK (natural killer)cell killing.

[0041] The methods further include administering to an animal or patienthaving a vascularized tumor at least a first pharmaceutical compositioncomprising an amount of at least a first antibody construct effective toinduce, or specifically induce, complement-mediated lysis of at least aportion of the tumor vascular endothelial cells. Herein, the firstantibody construct is a naked or unconjugated antibody, or effectivefragment thereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, present, expressed,translocated, presented or complexed at the luminal surface of tumorvascular endothelial cells and that induces complement-mediated lysis ofat least a portion of the tumor vascular endothelial cells, as opposedto endothelial cells in normal vessels. As used herein,“complement-mediated or complement-dependent lysis or cytotoxicity”means the process by which the complement-dependent coagulation cascadeis activated, multi-component complexes are assembled, ultimatelygenerating a lytic complex that has direct lytic action, causing cellpermeabilization. Anti-aminoplhospholipid antibodies for use in inducingcomplement-mediated lysis will generally include the Fc portion of theantibody. The complement-based mechanisms by which the present inventionmay operate further include “complement-activated ADCC”. In suchaspects, the administered naked or unconjugated antibodies, or fragmentsthereof, bind to an aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, present, expressed, translocated, presented orcomplexed at the luminal surface of tumor vascular endothelial cells andinduce complement-activated ADCC of at least a portion of the tumorvascular endothelial cells, as opposed to endothelial cells in normalvessels. “Complement-activated ADCC” is used to refer to the process bywhich complement, not an antibody Fc portion per se, holds amulti-component complex together and in which cells such as neutrophilslyse the target cell.

[0042] In other embodiments, the methods include administering to ananimal or patient having a vascularized tumor at least a firstpharmaceutical composition comprising an amount of at least a firstantibody construct effective to induce, or specifically induce,apoptosis in at least a portion of the tumor vascular endothelial cells.Herein, the first antibody construct is a naked or unconjugatedantibody, or antigen-binding fragment thereof, that binds to anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, present, expressed, translocated, presented orcomplexed at the luminal surface of tumor vascular endothelial cells andthat induces apoptosis in least a portion of the tumor vascularendothelial cells, as opposed to endothelial cells in normal vessels. Asused herein, “induces apoptosis” means induces the process of programmedcell death that, during the initial stages, maintains the integrity ofthe cell membrane, yet transmits the death-inducing signals into thecell. This is opposed to the mechanisms of cell necrosis, during whichthe cell membrane loses its integrity and becomes leaky at the onset ofthe process.

[0043] The anti-aminophospholipid antibody treatment methods willgenerally involve the administration of at least one dose of thepharmaceutically effective composition to the animal systemically, suchas via intravenous injection. However, any route of administration thatallows the antibody localize to the tumor vascular endothelial cells andto induce cell-mediated cytotoxicity, complement-mediated lysis and/orapoptosis thereof will be acceptable.

[0044] “Administration”, as used herein, therefore means provision ordelivery of anti-aminophospholipid antibodies in an amount(s) and for aperiod of time(s) effective to allow binding to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof blood transporting vessels of the vascularized tumor, and to exert atumor vasculature destructive and tumor-regressive effect. The passiveadministration of proteinaceous antibodies is generally preferred, inpart, for its simplicity and reproducibility.

[0045] However, the term “administration” is herein used to refer to anyand all means by which anti-aminophospholipid antibodies are deliveredor otherwise provided to the tumor vasculature. “Administration”therefore includes the provision of cells, such as hybridomas, thatproduce the anti-aminophospholipid antibodies in a manner effective toresult in the delivery of the anti-aminophospholipid antibodies to thetumor vasculature, and/or their localization to such vasculature. Insuch embodiments, it may be desirable to formulate or package theantibody-producing cells in a selectively permeable membrane, structureor implantable device, generally one that can be removed to ceasetherapy.

[0046] “Antibody administration”, as used herein, also extends to allmethods by which anti-aminophospholipid antibodies are generated in apatient (“endogenous antibodies”), allowing them to circulate andlocalize to the tumor vasculature. Therefore, “administration” alsoincludes the active immunization of a patient with an immunogenicallyeffective amount of an aminophospholipid sample, antigen or immunogen.All methods of human immunization are appropriate for use in suchembodiments, as exemplified by those described below in the context ofgenerating an anti-aminophospholipid antibody response in an animal inorder to obtain the antibody therefrom. Exogenous antibodyadministrationwill still generallybe preferred over cell andimmunization-based delivery, as this represents a less invasive methodthat allows the dose to be closely monitored and controlled.

[0047] The “antibody administration methods” of the invention alsoextend to the provision of nucleic acids that encodeanti-aminophospholipid antibodies in a manner effective to result in theexpression of the anti-aminophospholipid antibodies in the vicinity ofthe tumor vasculature, and/or in the expression ofanti-aminophospholipid antibodies that can localize to the tumorvasculature. Any gene therapy technique may be employed, such as nakedDNA delivery, recombinant genes and vectors, cell-based delivery,including ex vivo manipulation of patients' cells, and the like.

[0048] One of the benefits of the present invention is thataminophospholipids, particularly phosphatidylserine andphosphatidylethanolamine, are generally expressed or availablethroughout the tumor vessels. Aminophospholipid expression onestablished, intratumoral blood vessels is advantageous as targeting anddestroying such vessels will rapidly lead to anti-tumor effects.However, so long as the administered anti-aminophospholipid antibodiesbind to at least a portion of the blood transporting vessels,significant anti-tumor effects will ensue. This will not beproblematical as aminophospholipids, such as phosphatidylserine andphosphatidylethanolamine, are expressed on the large, central vessels,and also on veins, venules, arteries, arterioles and blood transportingcapillaries in all regions of the tumor.

[0049] In any event, the ability of the anti-arninophospholipidantibodies to destroy the tumor vasculature means that tumor regressioncan be achieved, rather than only tumor stasis. Tumor stasis is mostoften the result of anti-angiogenic therapies that target only thebudding vessels at the periphery of a solid tumor and stop the vesselsproliferating. Even if the present invention targeted more of theperipheral regions of the tumor in certain tumor types, which is notcurrently believed to be the case, destruction of the blood transportingvessels in such areas would still lead to widespread thrombosis andtumor necrosis.

[0050] In any of the foregoing methods, the terms anti-aminophospholipid“antibody, naked antibody and unconjugated antibody”, as used herein,refer broadly to any immunologic binding agent, such as polyclonal andmonoclonal IgG, IgM, IgA, IgD and IgE antibodies. Generally, IgG and/orIgM are preferred because they are the most common antibodies in thephysiological situation and because they are most easily made in alaboratory setting.

[0051] Polyclonal anti-aminophospholipid antibodies, obtained fromantisera, may be employed in the invention. However, the use ofmonoclonal anti-aminophospholipid antibodies (MAbs) will generally bepreferred. MAbs are recognized to have certain advantages, e.g.,reproducibility and large-scale production, that makes them suitable forclinical treatment. The invention thus provides monoclonal antibodies ofthe murine, human, monkey, rat, hamster, rabbit and even frog or chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will be used in certainembodiments.

[0052] As will be understood by those in the art, the immunologicbinding reagents encompassed by the term “anti-aminophospholipidantibody” extend to all naked and unconjugated anti-aminophospholipidantibodies from all species, and antigen binding fragments thereof,including dimeric, trimeric and multimeric antibodies; bispecificantibodies; chimeric antibodies; human and humanized antibodies;recombinant and engineered antibodies, and fragments thereof.

[0053] The term “anti-aminophospholipid antibody” is thus used to referto any anti-aminophospholipid antibody-like molecule that has an antigenbinding region, and includes antibody fragments such as Fab′, Fab,F(ab′)₂, single domain antibodies (DABs), Fv, scFv (single chain Fv),and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art.

[0054] In certain embodiments, the antibodies employed will be“humanized” or human antibodies. “Humanized” antibodies are generallychimeric monoclonal antibodies from mouse, rat, or other non-humanspecies, bearing human constant and/or variable region domains(“part-human chimeric antibodies”). Mostly, humanized monoclonalantibodies for use in the present invention will be chimeric antibodieswherein at least a first antigen binding region, or complementaritydetermining region (CDR), of a mouse, rat or other non-humananti-aminophospholipid monoclonal antibody is operatively attached to,or “grafted” onto, a human antibody constant region or “framework”.“Humanized” monoclonal antibodies for use herein may also beanti-aminophospholipid monoclonal antibodies from non-human specieswherein one or more selected amino acids have been exchanged for aminoacids more commonly observed in human antibodies. This can be readilyachieved through the use of routine recombinant technology, particularlysite-specific mutagenesis.

[0055] Entirely human, rather than “humanized”, anti-aminophospholipidantibodies may also be prepared and used in the present invention. Suchhuman antibodies may be polyclonal antibodies, as obtained from humanpatients that have any one or more of a variety of diseases, disordersor clinical conditions associated with the production ofanti-aminophospholipid antibodies. Such antibodies may be concentrated,partially purified or substantially purified for use herein.

[0056] A range of techniques are also available for preparing humanmonoclonal antibodies. As human patients with anti-aminophospholipidantibody-producing diseases exist, the anti-aminophospholipidantibody-producing cells from such patients may be obtained andmanipulated in vitro to provide a human monoclonal antibody. The invitro manipuiations or techniques include fusing to prepare a monoclonalantibody-producing hybridoma, and/or cloning the gene(s) encoding theanti-aminophospholipid antibody from the cells (“recombinant humanantibodies”).

[0057] Human anti-aminophospholipid antibody-producing cells may also beobtained from human subjects without an anti-aminophospholipidantibody-associated disease, i.e. “healthy subjects” in the context ofthe present invention. To achieve this, one would simply obtain apopulation of mixed peripheral blood lymphocytes from a human subject,including antigen-presenting and antibody-producing cells, and stimulatethe cell population in vitro by admixing with an immunogenicallyeffective amount of an aminophospholipid sample. Again, the humananti-aminophospholipid antibody-producing cells, once obtained, could beused in hybridoma and/or recombinant antibody production.

[0058] Further techniques for human monoclonal antibody productioninclude immunizing a transgenic animal, preferably a transgenic mouse,that comprises a human antibody library with an immunogenicallyeffective amount of an aminophospholipid sample. This also generateshuman anti-aminophospholipid antibody-producing cells for furthermanipulation in hybridoma and/or recombinant antibody production, withthe advantage that spleen cells, rather than peripheral blood cells, canbe readily obtained from the transgenic animal or mouse.

[0059] As used herein, the term “anti-aminophospholipid antibody” isused co-extensively with “naked and unconjugated” to meananti-aminophospholipid antibodies, and antigen binding fragmentsthereof, that are not conjugated to, or operatively associated with, aneffector molecule, such as a cytotoxic agent or coagulant. In additionto non-effector modifications of the antibody, and in vivo interactions,the term “naked” in no way excludes combinations of the antibody withother therapeutic agents, as disclosed in detail herein.

[0060] In contrast, anti-aminophospholipid antibodies, bispecificantibodies, and antigen binding fragments thereof, that are conjugatedto, or operatively associated with, cytotoxic or anticellular agents arereferred to herein as “anti-aminophospholipid immunotoxins”. Likewise,anti-aminophospholipid antibodies, bispecific antibodies, and antigenbinding fragments thereof, that are conjugated to, or operativelyassociated with, coagulation factors are herein referred to as“anti-aminophospholipid coaguligands”. The use of anti-aminophospholipidimiunotoxins and anti-aminophospholipid coaguligands in tumor treatmentis also contemplated by the present inventors and is disclosed andclaimed in first and second provisional applications Ser. Nos.60/092,589 (filed Jul. 13, 1998) and 60/110,600 (filed Dec. 2, 1998) andin co-filed U.S. and PCT patent applications (Attorney Docket Nos.4001.002300, 4001.002382, 4001.002383 and 4001.002210), eachspecifically incorporated herein by reference.

[0061] Preferred anti-aminophospholipid antibodies for use in thepresent invention are anti-phosphatidylserine (anti-PS) andanti-plhosphatidylethanolaninie (anti-PE) antibodies. Anti-PS antibodieswill generally recognize, bind to or have immunospecificity for the PSmolecule present, expressed, translocated, presented or complexed at theluminal surface of tumor vascular endothelial cells. Suitable antibodieswill thus bind to phosphatidyl-L-serine (Umeda et al., 1989;incorporated herein by reference). Anti-PE antibodies will generallyrecognize, bind to or have immunospecificity for the PE moleculepresent, expressed, translocated, presented or complexed at the luminalsurface of tumor vascular endothelial cells.

[0062] Administering anti-aminophospholipid antibodies to an animal witha tumor will result in specific binding of the antibody to theaminophospholipid molecules present, expressed or translocated to theluminal surface of the tumor blood vessels, i.e., the antibodies willbind to the aminophospholipid molecules in a natural, biologicalenvironment. Therefore, no particular manipulation will be necessary toensure antibody binding.

[0063] However, it is of scientific interest to note thataminophospholipids may be most frequently recognized, or bound, byanti-aminophospholipid antibodies when the aminophospholipid moleculesare associated with one or more proteins or other non-lipid biologicalcomponents. For example, anti-PS antibodies that occur as a sub-set ofanti-phospholipid (anti-PL) antibodies in patients with certain diseasesand disorders are now believed to bind to PS in combination withproteins such as β₂-glycoprotein I (β₂-GPI or apolipoprotein H, apoH)and prothrombin (U.S. Pat. No. 5,344,758; Rote, 1996; each incorporatedherein by reference). Similarly, anti-PE antibodies that occur indisease states are now believed to bind to PE in combination withproteins such as low and high molecular weight kininogen (HK),prekallikrein and even factor XI (Sugi and McIntyre, 1995; 1996a; 1996b;each incorporated herein by reference).

[0064] This is the meaning of the terms “presented” and “complexed at”the luminal surface of tumor blood vessels, as used herein, which meanthat the aminophospholipid molecules are present at the surface of tumorblood vessels in an antibody-binding competent state, irrespective ofthe molecular definition of that particular state. PS may even betargeted as a complex with factor II/IIa, VII/VIIa, IX/IXa and X/Xa.Moreover, the nature of the aminophospholipid target may change duringpractice of thc invention, as the initial aminophospholipid antibodybinding, anti-endothelial cell and anti-tumor effects may result inbiological changes that alter the number, conformation and/or type ofthe aminophospholipid target epitope(s).

[0065] The term “anti-aminophospholipid antibody”, as used in thecontext of the present invention, therefore means any naked orunconjugated anti-aminophospholipid antibody, immunological bindingagent or antisera; monoclonal, human, humanized, dimeric, trimeric,multimeric, chimeric, bispecific, recombinant or engineered antibody; orFab′, Fab, F(ab′)₂, DABs, Fv or scFv antigen binding fragment thereof;that at least binds to a lipid and amino group-containing complex oraminophospholipid target, preferably a phosphatidylserine- orphosphatidylethanolamine-based target.

[0066] The requirement that the antibody “at least bind to anaminophospholipid target” is met by the antibody binding to any and/orall physiologically relevant forms of aminophospholipids, includingso-called “hexagonal” and “hexagonal phase II” PS and PE (HexII PS andHexII PE) (Rauch et al., 1986; Rauch and Janoff, 1990; Berard et al,1993; each incorporated herein by reference) and PS and PE incombination with any other protein, lipid, membrane component, plasma orserum component, or any combination thereof. Thus, an“anti-aminophospholipid antibody” is an antibody that binds to anaminophospholipid in the tumor blood vessels, notwithstanding the factthat bilayer or micelle aminophospholipids may be considered to beimmunogenically neutral.

[0067] The anti-aminophospholipid antibodies may recognize, bind to orhave immunospecificity for aminophospholipid molecules, or animmunogenic complex thereof (including hexagonal aminophospholipids andprotein combinations), to the exclusion of other phospholipids orlipids. Such antibodies may be termed “aminophospholipid-specific oraminophospholipid-restricted antibodies”, and their use in the inventionwill often be preferred. “Aminophospholipid-specific or aminophospholipid-restricted antibodies” will generally exhibitsignificant binding to aminophosplholipids, while exhibiting little orno significant binding to other lipid components, such asphosphatidylinositol (PI), phosphatidylglyccrol (PG) and evenphosphatidylcholine (PC) in certain embodiments.

[0068] “PS-specific or PS-restricted antibodies” will generally exhibitsignificant binding to PS, while exhibiting little or no significantbinding to lipid components such as phosphatidylethanolamine andcardiolipin (CL), as well as PC, PI and PG. “PE-specific orPE-restricted antibodies” will generally exhibit significant binding toPE, while exhibiting little or no significant binding to lipidcomponents such as phosphatidylserine and cardiolipin, as well as PC, PIand PG. The preparation of specific anti-aminophospholipid antibodies isreadily achieved, e.g., as disclosed by Rauch et al. (1986); Umeda etal. (1989); Rauch and Janoff (1990); and Rote et al. (1993); eachincorporated herein by reference.

[0069] “Cross-reactive anti-aminophospholipid antibodies” thatrecognize, bind to or have immunospecificity for an aminophospholipidmolecule, or an immunogenic complex thereof (including hexagonalaminophospholipids and protein combinations), in addition to exhibitinglesser but detectable binding to other phospholipid or lipid componentsare by no means excluded from use in the invention. Such “cross-reactiveanti-aminophospholipid antibodies” may be employed so long as they bindto an aminophospholipid present, expressed, translocated, presented orcomplexed at the luminal surface of tumor vascular endothelial cells andexert an anti-tumor effect upon administration in vivo.

[0070] Further suitable aminophospholipid-specific oraminophospholipid-restricted antibodies are those anti-aminophospholipidantibodies that bind to both PS and PE. While clearly being specific orrestricted to aminophospholipids, as opposed to other lipid components,antibodies exist that bind to each of the preferred targets of thepresent invention. Examples of such antibodies for use in the inventioninclude, but are not limited to, PS3A, PSF6, PSF7, PSB4, PS3H1 andPS3E10 (Igarashi et al., 1991; incorporated herein by reference)

[0071] Further exemplary anti-PS antibodies for use in the inventioninclude, but are not limited to BA3B5C4, PS4A7, PS1G3 and 3SB9b; withPS4A7, PS1G3 and 3SB9b generally being preferred. Monoclonal antibodies,humanized antibodies and/or antigen-binding fragments based upon the3SB9b antibody (Rote el al., 1993; incorporated herein by reference) arecurrently most preferred.

[0072] Although aminophospholipids, such as PS and PE, in bilayer ormicelle form have been reported to be non- or weakly antigenic, or non-or weakly-immunogenic, the scientific literature has reported nodifficulties in generating anti-aminophospholipid antibodies, such asanti-PS and anti-PE antibodies. Anti-aminophospholipid antibodies ormonoclonal antibodies may therefore be readily prepared by preparativeprocesses and methods that comprise:

[0073] (a) preparing an anti-aminophospholipidantibody-producingcell;and

[0074] (b) obtaining an anti-aminophospholipid antibody or monoclonalantibody from the antibody-producing cell.

[0075] The processes of preparinganti-aminophospholipidantibody-producingcells and obtaininganti-aminophospholipidantibodies therefrom may be conduced in situ in agiven patient. That is, simply providing an immunogenically effectiveamount of an immunogenic aminophospholipid sample to a patient willresult in anti-aminophospholipid antibody generation. Thus, theanti-aminophospholipid antibody is still “obtained” from theantibody-producing cell, but it does not have to be isolated away from ahost and subsequently provided to a patient, being able to spontaneouslylocalize to the tumor vasculature and exert its biological anti-tumoreffects.

[0076] As disclosed herein, anti-aminophospholipid antibody-producingcells may be obtained, and antibodies subsequently isolated and/orpurified, from human patients with anti-aminophospholipidantibody-producing diseases, from stimulating peripheral bloodlymphocytes with aminophospholipidsin vitro, and also by immunizationprocesses and methods. The latter of which generally comprise:

[0077] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of an immunogenicallyeffective amount of an immunogenic aminophospholipid sample (such as ahexagonal, or hexagonal phase II form of an aminophospholipid),preferably an immunogenic PS or PE sample; and

[0078] (b) obtaining an anti-aminophospholipid antibody-producing cellfrom the immunized animal.

[0079] The immunogenically effective amount of the aminophospholipidsample or samples may be a Salmonella-coated aminophospholipid sample(Umeda et al, 1989; incorporated herein by reference); anaminophospholipidmicelle, liposome, lipid complex or lipid formulationsample; or an aminophospholipidsample fabricated with SDS. Any suchaminophospholipidsample may be administered in combination with anysuitable adjuvant, such as Freund's complete adjuvant (Rote et al, 1993;incorporated herein by reference). Any empirical technique or variationmay be employed to increase immunogenicity, and/or hexagonal orhexagonal phase II forms of the aminophospholipids may be administered.

[0080] The immunization may be based upon one or more intrasplenicinjections of an immunogenically effective amount of anaminophospholipid sample (Umeda et al., 1989; incorporated herein byreference).

[0081] Irrespective of the nature of the immunization process, or thetype of immunized animal, anti-aminophospholipid antibody-producingcells are obtained from the immunized animal and, preferably, furthermanipulated by the hand of man. “An immunized animal”, as used herein,is a non-human animal, unless otherwise expressly stated. Although anyantibody-producing cell may be used, most preferably, spleen cells areobtained as the source of the antibody-producing cells. Theanti-aminophospholipid antibody-producing cells may be used in apreparative process that comprises:

[0082] (a) tusisng an anti-am inopliospholipidantibody-producingcellwith an immortal cell to prepare a hybridoma that produces ananti-aminophospliolipidmonoclonal antibody and

[0083] (b) obtaining an anti-aminophospholipidmonoclonal antibody fromthe hybridoma.

[0084] Hybridoma-based monoclonal antibody preparative methods thusinclude those that comprise:

[0085] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of an immunogenicallyeffective amount of an immunogenic aminophospholipid sample (such as ahexagonal, or hexagonal phase II form of an aminophospholipid),preferably an immunogenic PS or PE sample;

[0086] (b) preparing a collection of monoclonal antibody-producinghybridomas from the immunized animal;

[0087] (c) selecting from the collection at least a first hybridoma thatproduces at least a first anti-aminophospholipid monoclonal antibody,and preferably, at least a first aminophospholipid-specific monoclonalantibody; and

[0088] (d) culturing the at least a firstanti-aminophospholipid-producing or aminophospholipid-specific hybridomato provide the at least a first anti-aminophospholipid monoclonalantibody or aminophospholipid-specific monoclonal antibody; andpreferably

[0089] (e) obtaining the at least a first anti-aminophospholipidmonoclonal antibody or aminophosplholipid-specific monoclonal antibodyfrom the cultured at least a first hybridoma.

[0090] As non-human animals are used for immunization, theanti-aminophospholipid monoclonal antibodies obtained from such ahybridoma will often have a non-human make up. Such antibodies may beoptionally subjected to a humanization process, grafting or mutation, asknown to those of skill in the art and further disclosed herein.Alternatively, transgenic animals, such as mice, may be used thatcomprise a human antibody gene library. Immunization of such animalswill therefore directly result in the generation of humananti-aminophospholipid antibodies.

[0091] After the production of a suitable antibody-producing cell, mostpreferably a hybridoma, whether producing human or non-human antibodies,the monoclonal antibody-encoding nucleic acids may be cloned to preparea “recombinant” monoclonal antibody. Any recombinant cloning techniquemay be utilized, including the use of PCR to prime the synthesis of theantibody-encoding nucleic acid sequences. Therefore, yet furtherappropriate monoclonal antibody preparative methods include those thatcomprise using the anti-aminophospholipid antibody-producing cells asfollows:

[0092] (a) obtaining at least a first anti-aminophospholipidantibody-encoding nucleic acid molecule or segment from ananti-aminophospholipid antibody-producing cell, preferably a hybridoma;and

[0093] (b) expressing the nucleic acid molecule or segment in arecombinant host cell to obtain a recombinantanti-aminophospholipidmonoclonal antibody.

[0094] However, other powerful recombinant techniques are available thatare ideally suited to the preparation of recombinant monoclonalantibodies. Such recombinant techniques include the phagemidlibrary-based monoclonal antibody preparative methods comprising:

[0095] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of an immunogenicallyeffective amount of an immunogenic aminophospholipid sample (such as ahexagonal, or hexagonal phase II form of an aminophospholipid),preferably an immunogenic PS or PE sample;

[0096] (b) preparing a combinatorial immunoglobulin phagemid libraryexpressing RNA isolated from the antibody-producing cells, preferablyfrom the spleen, of the immunized animal;

[0097] (c) selecting from the phagemid library at least a first clonethat expresses at least a first anti-aminophospholipid antibody, andpreferably, at least a first aminophospholipid-specific antibody;

[0098] (d) obtaining anti-aminophospholipidantibody-encodingnucleicacids from the at least a first selected clone and expressing thenucleic acids in a recombinant host cell to provide the at least a firstanti-aminophospholipid antibody or aminophospholipid-specific antibody;and preferably

[0099] (e) obtaining the at least a first anti-aminophospholipidantibody or aminophospholipid-specific antibody expressed by the nucleicacids obtained from the at least a first selected clone.

[0100] Again, in such phagemid library-based techniques, transgenicanimals bearing human antibody gene libraries may be employed, thusyielding recombinant human monoclonal antibodies.

[0101] Irrespective of the manner of preparation of a firstanti-aminophospholipid antibody nucleic acid segment, further suitableanti-aminophospholipid antibody nucleic acid segments may be readilyprepared by standard molecular biological techniques. In order toconfirm that any variant, mutant or second generationanti-aminophospholipid antibody nucleic acid segment is suitable for usein the present invention the nucleic acid segment will be tested toconfirm expression of an antibody that binds to an aminiophospholipid.Preferably, the variant, mutant or second generationanti-aminophospholipid antibody nucleic acid segment will also be testedto confirm hybridization to an anti-aminophosplholipid antibody nucleicacid segment under standard, more preferably, standard stringenthybridization conditions. Exemplary suitable hybridization conditionsinclude hybridization in about 7% sodium dodecyl sulfate (SDS), about0.5 M NaPO₄, about 1 mM EDTA at about 50° C.; and washing with about 1%SDS at about 42° C.

[0102] As a variety of recombinant monoclonal antibodies, whether humanor non-human in origin, may be readily prepared, the treatment methodsof the invention may be executed by providing to the animal or patientat least a first nucleic acid segment that expresses a biologicallyeffective amount of at least a first anti-aminophospholipid antibody inthe patient. The “nucleic acid segment that expresses ananti-aminophospholipid antibody” will generally be in the form of atleast an expression construct, and may be in the form of an expressionconstruct comprised within a virus or within a recombinant host cell.Preferred gene therapy vectors of the present invention will generallybe viral vectors, such as comprised within a recombinant retrovirus,herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV),cytomegalovirus (CMV), and the like.

[0103] In certain embodiments, the vasculature of the vascularized tumorof the animal or patient to be treated may be first imaged. Generallythis is achieved by first administering to the animal or patient adiagnostically effective amount of at least a first pharmaceuticalcomposition comprising at least a first detectably-labeledaminophospholipid binding construct, such as an anti-aminophospholipidantibody-detectable agent construct, that binds to and identifies anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, present, expressed, translocated, presented orcomplexed at the luminal surface of blood vessels of the vascularizedtumor. The invention thus further provides compositions for use in, andmethods of, distinguishing between tumor and/or intratumoral bloodvessels and normal blood vessels.

[0104] The “distinguishing” is achieved by administering one or more ofthe detectably-labeled ami nophospholipid binding constructs described.The detectably-labeled aminophospholipid binding construct oranti-aminophospholipid antibody-detectable agent construct may comprisean X-ray detectable compound, such as bismuth (III), gold (III),lanthanum (III) or lead (II); a radioactive ion, such as copper⁶⁷,gallium⁶⁷, gallium⁶¹, indium¹¹³, indium¹¹³, iodine¹²³, iodine¹²⁵,iodine¹³¹, mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷,rubidium¹⁰³, technetium^(99m) or yttrium⁹⁰; a nuclear magneticspin-resonance isotope, such as cobalt (II), copper (II), chromium(III), dysprosium (III), erbium (III), gadolinium (III), holmium (III),iron (II), iron (III), manganese (II), neodymium (III), nickel (II),samarium (III), terbium (III), vanadium (II) or ytterbium (III); orrhodamine or fluorescein.

[0105] Pre-imaging before tumor treatment may thus be carried out by:

[0106] (a) administering to the animal or patient a diagnosticallyeffective amount of a pharmaceutical composition comprising at least afirst detectably-labeled aminophospholipid binding construct thatcomprises a diagnostic agent operatively attached to an antibody,binding protein or ligand, or aminophospholipid binding fragmentthereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, present, expressed,translocated, presented or complexed at the luminal surface of bloodvessels or intratumoral blood vessels of the vascularized tumor; and

[0107] (b) subsequently detecting the detectably-labeledaminophospholipid binding construct bound to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, on theluminal surface of tumor or intratumoral blood vessels, therebyobtaining an image of the tumor vasculature.

[0108] Cancer treatment may also be carried out by:

[0109] (a) forming an image of a vascularized tumor by administering toan animal or patient having a vascularized tumor a diagnosticallyminimal amount of at least a first detectably-labeled aminophospholipidbinding construct comprising a diagnostic agent operatively attached toan antibody, binding protein or ligand, or aminophospholipid bindingfragment thereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, on the luminal surfaceof tumor or intratumoral blood vessels of the vascularized tumor,thereby forming a detectable image of the tumor vasculature; and

[0110] (b) subsequently administering to the same animal or patient atherapeutically optimized amount of at least a first naked antibody, orantigen-binding fragment thereof, that binds to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, on the tumorblood vessel luminal surface and thereby destroys the tumor vasculature.

[0111] Imaging and treatment formulations or medicaments are thusprovided, which generally comprise:

[0112] (a) a first pharmaceutical composition comprising adiagnostically effective amount of a detectably-labeledaminophospholipid binding construct that comprises a detectable agentoperatively attached to an antibody, binding protein or ligand, oraminophospholipidbinding fragment thereof, that binds to anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, on the luminal surface of tumor orintratumoral blood vessels of the vascularized tumor; and

[0113] (b) at least a second pharmaceutical composition comprising atherapeutically effective amount of at least a second, unconjugatedanti-aminophospholipid antibody, preferably anti-phosphatidylserine oranti-pholsplatidylethallolamine, or antigen binding fragment thereof.

[0114] In such methods and medicaments, advantages will be realizedwherein the first and second pharmaceutical compositions comprise thesame targeting agents, e.g., anti-aminophospholipid antibodies, orfragments thereof, from the same antibody preparation, or preferably,from the same antibody-producing hybridoma.

[0115] In the vasculature imaging aspects of the invention, it isrecognized that the administered detectably-labeled aminophospholipidbinding construct, or anti-aminophospholipid antibody-detectable agent,may itself have a therapeutic effect. Whilst this would not be excludedfrom the invention, the amounts of the detectably-labeledconstructs tobe administered would generally be chosen as “diagnostically effectiveamounts”, which are typically lower than the amounts required fortherapeutic benefit.

[0116] In the imaging embodiments, the targeting agent may be eitherantibody-based or binding ligand- or binding protein-based. Although notpreviously connected with tumors or tumor vasculature, detectablylabeled aminophospholipid binding ligand compositions are known in theart and can now, in light of this motivation and the present disclosure,be used in the combined imaging and treatment aspects of the presentinvention. The detectably-labeled annexins of U.S. Pat. No. 5,627,036;WO 95/19791; WO 95/27903; WO 95/34315; WO 96/17618; and WO 98/04294;each incorporated herein by reference; may thus be employed.

[0117] The foregoing imaging and treatment formulations or medicamentsmay also further comprise one or more anti-cancer agents. That is, thepresent invention encompasses imaging and combination treatmentformulations and medicaments that generally comprise (a) diagnosticallyeffective amounts of detectably-labeled aminophospholipid bindingconstructs; (b) therapeutically effective amounts of unconjugatedanti-aminophospholipid antibodies, preferably anti-phosphatidylserine oranti-phosphatidylethanolamine, or antigen binding fragments thereof; and(c) therapeutically effective amounts of at least other anti-canceragent(s).

[0118] In still further embodiments, the animals or patients to betreated by the present invention are further subjected to surgery orradiotherapy, or are provided with a therapeutically effective amount ofat least a first anti-cancer agent. The “at least a first anti-canceragent” in this context means “at least a first anti-cancer agent inaddition to the naked anti-aminophospholipid antibody” (preferablyanti-phosphatidylserine or anti-phosphatidylethanolamine). The “at leasta first anti-cancer agent” may thus be considered to be “at least asecond anti-cancer agent”, where the naked anti-aminophospholipidantibody is a first anti-cancer agent. However, this is purely a matterof semantics, and the practical meaning will be clear to those ofordinary skill in the art.

[0119] The at least a first anti-cancer agent may be administered to theanimal or patient substantially simultaneously with theanti-aminophospholipid antibody, or antigen-binding fragment thereof;such as from a single pharmaceutical composition or from twopharmaceutical compositions administered closely together.

[0120] Alternatively, the at least a first anti-cancer agent may beadministered to the animal or patient at a time sequential to theadministration of the at least a first anti-aminophospholipid antibody,or antigen-binding fragment thereof. “At a time sequential”, as usedherein, means “staggered”, such that the at least a first anti-canceragent is administered to the animal or patient at a time distinct to theadministration of the at least a first anti-aminophospholipid antibody.Generally, the two agents are administered at times effectively spacedapart to allow the two agents to exert their respective therapeuticeffects, i.e., they are administered at “biologically effective timeintervals”.

[0121] The at least a first anti-cancer agent may be administered to theanimal or patient at a biologically effective time prior to theanti-aminophospholipid antibody or fragment thereof, or at abiologically effective time subsequent to the anti-aminophospholipidantibody fragment. Administration of one or more non-aminophospholipidtargeted anti-cancer agents at a therapeutically effective timesubsequent to an anti-aminophospholipid antibody may be particularlydesired wherein the anti-cancer agent is an anti-tumor cell immunotoxindesigned to kill tumor cells at the outermost rim ot the tumor, and/orwherein the anti-cancer agent is an anti-angiogenic agent designed toprevent micrometastasis of any remaining tumor cells. Suchconsiderations will be known to those of skill in the art.

[0122] Administration of one or more non-aminophospholipid targetedanti-cancer agents at a therapeutically effective time prior to ananti-aminophospholipid antibody may be particularly employed where theanti-cancer agent is designed to increase aminophospholipid expression.This may be achieved by using anti-cancer agents that injure, or induceapoptosis in, the tumor endothelium. Exemplary anti-cancer agentinclude, e.g., taxol, vincristine, vinblastine, neomycin,combretastatin(s), podophyllotoxin(s), TNF-α, angiostatin, endostatin,vasculostatin, α_(v)β₃ antagonists, calcium ionophores, calcium-fluxinducing agents, any derivative or prodrug thereof.

[0123] The one or more additional anti-cancer agents may bechemotherapeutic agents, radiotherapeutic agents, cytokines,anti-angiogenic agents, apoptosis-inducing agents or anti-cancerimmunotoxins or coaguligands. “Chemotherapeutic agents”, as used herein,refer to classical chemotherapeutic agents or drugs used in thetreatment of malignancies. This term is used for simplicitynotwithstanding the fact that other compounds may be technicallydescribed as chemotherapeutic agents in that they exert an anti-cancereffect. However, “chemotherapeutic” has come to have a distinct meaningin the art and is being used according to this standard meaning.

[0124] A number of exemplary chemotherapeutic agents are describedherein. Those of ordinary skill in the art will readily understand theuses and appropriate doses of chemotherapeutic agents, although thedoses may well be reduced when used in combination with the presentinvention. A new class of drugs that may also be termed“chemotherapeutic agents” are agents that induce apoptosis. Any one ormore of such drugs, including genes, vectors and antisense constructs,as appropriate, may also be used in conjunction with the presentinvention.

[0125] Anti-cancer immunotoxins or coaguligands are further appropriateanti-cancer agents. “Anti-cancer immunotoxins or coaguligands”, ortargeting-agent/therapeutic agent constructs, are based upon targetingagents, including antibodies or antigen binding fragments thereof, thatbind to a targetable component of a tumor cell, tumor vasculaturc ortumor stroma, and that are operatively attached to a therapeutic agent,generally a cytotoxic agent (immunotoxin) or coagulation factor(coaguligand). A “targetable component” of a tumor cell, tumorvasculature or tumor stroma, is preferably a surface-expressed,surface-accessible or surface-localized component, although componentsreleased from necrotic or otherwise damaged tumor cells or vascularendothelial cells may also be targeted, including cytosolic and/ornuclear tumor cell antigens.

[0126] Both antibody and non-antibody targeting agents may be used,including growth factors, such as VEGF and FGF; peptides containing thetripeptide R-G-D, that bind specifically to the tumor vasculature; andother targeting components such as annexins and related ligands.

[0127] At-tumor cell immunotoxins or coaguligands may compriseantibodies exemplified by the group consisting of B3 (ATCC HB 10573)),260F9 (ATCC HB 8488), D612 (ATCC HB 9796) and KS1/4, said KS1/4 antibodyobtained from a cell comprising the vector pGKC2310 (NRRL B-118356) orthe vector pG2A52 (NRRL B-18357).

[0128] Anti-tumor stroma imimunotoxins or coaguligands will generallycomprise antibodies that bind to a connective tissue component, abasement membrane component or an activated platelet component; asexemplified by binding to fibrin, RIBS or LIBS.

[0129] Anti-tumor vasculature immunotoxins or coaguligands may compriseligands, antibodies, or fragments thereof, that bind to asurface-expressed, surface-accessible or surface-localized component ofthe blood transporting vessels, preferably the intratumoral bloodvessels, of a vascularized tumor. Such antibodies include those thatbind to surface-expressed components of intratumoral blood vessels of avascularized tumor, including aminophospholipids themselves, andintratumoral vasculature cell surface receptors, such as endoglin (TEC-4and TEC-11 antibodies), a TGFβ receptor, E-selectin, P-selectin, VCAM-1,ICAM-1 PSMA, a VEGF/VPF receptor, an FGF receptor, a TIE, α_(v)β₃integrin, plelotropin, endosialin and MHC Class II proteins. Theantibodies may also bind to cytokine-inducible or coagulanit-induciblecomponents of intratumoral blood vessels.

[0130] Other anti-tumor vasculature immunotoxins or coaguligands maycomprise antibodies, or fragments thereof, that bind to a ligand orgrowth factor that binds to an intratumoral vasculature cell surfacereceptor. Such antibodies include those that bind to VEGF/VPF (GV39 andGV97 antibodies), FGF, TGFβ, a ligand that binds to a TIE, atumor-associated fibronectin isoform, scatter factor/hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF and TIMP. The antibodies, orfragments thereof, may also bind to a ligand:receptor complex or agrowth factor:receptor complex, but not to the ligand or growth factor,or to the receptor, when the ligand or growth factor or the receptor isnot in the ligand:receptor or growth factor:receptor complex.

[0131] Anti-tumor cell, anti-tumor stroma or anti-tumor vasculatureantibody-therapeutic agent constructs may comprise anticellular,cytostatic or cytotoxic agents such as plant-, fuingus- orbacteria-derived toxins (immunotoxins). Ricin A chain and deglycosylatedricin A chain will often be preferred, and gelonin and angiopoletins arealso contemplated. Anti-tumor cell, anti-tumor stroma or anti-tumorvasculature antibody-therapeutic agent constructs may comprisecoagulation factors or second antibody binding regions that bind tocoagulation factors (coaguligands). The operative association withTissue Factor or Tissue Factor derivatives, such as truncated TissueFactor, will often be preferred.

[0132] The present invention yet further provides a series of noveltherapeutic kits, medicaments and/or cocktails for use in conjunctionwith the methods of the invention. The kits, medicaments and/orcocktails generally comprise a combined effective amount of ananti-cancer agent and an antibody, or an antigen-binding fragmentthereof, that binds to an aminophospholipid,preferablyphosphatidylserine or phosphatidylethanolamine.

[0133] Where the primary purpose of a kit of the invention is incombination therapy, the kit may nonetheless still further comprise animaging component, generally a diagnostically effective amount of adetectably-labeled aminophospholipid binding construct, such as alabeled anti-aminophospholipid antibody or antigen binding fragmentthereof.

[0134] The kits and medicaments will comprise, preferably in suitablecontainer means, a biologically effective amount of at least a firstantibody, or an antigen-binding fragment thereof, that binds to anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine; in combination with a biologically effectiveamount of at least a first anti-cancer agent. The components of the kitsand medicaments may be comprised within a single container or containermeans, or comprised within distinct containers or container means. Thecocktails will generally be admixed together for combined use.

[0135] The entire range of anti-aminophospholipid antibodies, asdescribed above, may be employed in the kits, medicaments and/orcocktails, with anti-PS, anti-PE, human, humanized and monoclonalantibodies, or fragments thereof, being preferred. The anti-canceragents are also those as described above, including chemotherapeuticagents, radiotherapeutic agents, anti-angiogenic agents, apoptopicagents, immunotoxins and coaguligands. Agents formulated for intravenousadministration will often be preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

[0136] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0137]FIG. 1A and FIG. 1B. Activity of cell-bound anti-VCAM-1·tTF invitro. FIG. 1A. Binding of anti-VCAM-1·tTF coaguligand to unstimulated(control) and IL-1α-activated bEnd.3 cells. FIG. 1B. Generation offactor Xa by cell-bound anti-VCAM-1·tTF coaguligand.

[0138]FIG. 2. Retardation of growth of L540 tumors in mice treated withanti-VCAM-1·tTF. L540 tumor bearing mice were injected i.v. with eithersaline, 20 μg of anti-VCAM-1·tTF, 4 μg of unconjugated tTF or 20 μg ofcontrol 1gG·tTF. Injections were repeated on day 4 and 8 after the firsttreatment. Tumors were measured daily. Mean tumor volume and SD of 8mice per group is shown.

[0139]FIG. 3. Annexin V blocks coaguligand activation of Factor X invitro. IL-1α-stimulated bEnd.3 cells were incubated with anti-VCAM·tTFcoaguligand in 96-well microtiter plates, as described in Example V.Annexin V was added at concentrations ranging from 0.1 to 10 μg/ml (asshown) and cells were incubated for 30 min. before addition of dilutedProplex T. The amount of Factor Xa generated in the presence or absenceof Annexin V was determined using a chromogenic substrate, as describedin Example V.

[0140]FIG. 4A and FIG. 4B. Anti-tumor effects of naked anti-PSantibodies in animals with syngeneic and xenogeneic tumors. 1×10⁷ cellsof murine colorectal carcinoma Colo 26 (FIG. 4A) or human Hodgkin'slymphoma L540 (FIG. 4B) were injected subcutaneously into the rightflank of Balb/c mice (FIG. 4A) or male CB17 SCID mice (FIG. 4B),respectively. Tumors were allowed to grow to a size of about 0.6-0.9 cm³and then the mice (4 animals per group) were injected i.p. with 20 μg ofnaked anti-PS antibody (open squares) or saline (open circles) (controlmouse IgM gave similar results to saline.). Treatment was repeated 3times with a 48 hour interval. Animals were monitored daily for tumormeasurements and body weight. Tumor volume was calculated as describedin Example VII. Mice were sacrificed when tumors had reached 2 cm³, orearlier if tumors showed signs of necrosis or ulceration.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0141] A. Tumor Destruction using VCAM-1 Coaguligand

[0142] Solid tumors and carcinomas account for more than 90% of allcancers in man. Although the use of monoclonal antibodies andimmunotoxins has been investigated in the therapy of lymphomas andleukemias (Vitetta et al., 1991), these agents have been disappointinglyineffective in clinical trials against carcinomas and other solid tumors(Abrams and Oldham, 1985). A principal reason for the ineffectiveness ofantibody-based treatments is that macromolecules are not readilytransported into solid tumors. Even once within a tumor mass, thesemolecules fail to distribute evenly due to the presence of tightJunctions between tumor cells, fibrous stroma, interstitial pressuregradients and binding site barriers (Dvorak el al., 1991).

[0143] In developing new strategies for treating solid tumors, themethods that involve targeting the vasculature of the tumor, rather thanthe tumor cells, offer distinct advantages. An effective destruction orblockade of the tumor vessels arrests blood flow through the tumor andresults in an avalanche of tumor cell death. Antibody-toxin andantibody-coagulant constructs have already been used to great effect inthe specific targeting and destruction of tumor vessels, resulting intumor necrosis (Burrows et al., 1992; Burrows and Thorpe, 1993; WO93/17715; WO 96/01653; Huang et al., 1997; each incorporated herein byreference).

[0144] Tumor vasculature-targeted cytotoxic agents are described in thefollowing patents and patent applications: U.S. Pat. Nos. 5,855,866;5,776,427; 5,863,538; and 5,660,827; and U.S. applications Ser. Nos.07/846,349; 08/295,868 (U.S. Pat. No. 5,___,___; Issue Fee paid);08/350,212 (U.S. Pat. No. 5,___,___; Issue Fee paid); and 08/457,869(Notice of Allowance Received); each incorporated herein by reference.Tumor targeted coagulants are described in the following patents andpatent applications: U.S. Pat. Nos. 5,855,866 and 5,877,289; U.S.applications Ser. Nos. 07/846,349; 08/350,212 (Patent No. 5,___,___;Issue Fee paid); 08/482,369 (U.S. Pat. No. 5,___,___; Issue Fee paid);08/487,427 (U.S. Pat. No. 5,___,___; Issue Fee paid); and 08/479,727(U.S. Pat. No. 5,___,___; Issue Fee paid); each incorporated herein byreference.

[0145] Where antibodies, growth factors or other binding ligands areused to specifically deliver a coagulant to the tumor vasculature, suchagents are termed “coaguligands”. A currently preferred coagulant foruse in coaguligands is truncated Tissue Factor (tTF) (Huang et al.,1997; WO 96/01653; U.S. Pat. No. 5,877,289. TF is the major initiator ofblood coagulation (Ruf et al., 1991). At sites of injury, FactorVII/VIIa in the blood comes into contact with, and binds to, TF on cellsin the perivascular tissues. The TF:VIIa complex, in the presence of thephospholipid surface, activates factors IX and X. This, in turn, leadsto the formation of thrombin and fibrin and, ultimately, a blood clot(Ruf′and Edgington, 1994).

[0146] The recombinant, truncated form of tissue factor (tTF), lackingthe cytosolic and transmembrane domains, is a soluble protein that hasabout five orders of magnitude lower coagulation inducing ability thannative TF (Stone e al., 1995; Huang el al., 1997). This is because TFneeds to be associated with phospholipids for the complex with Vlla toactivate IXa or Xa efficiently. However, when tTF is delivered to tumorvascular endothelium by means of a targeting antibody or agent, it isbrought back into proximity to a lipid surface and regains thrombogenicactivity (Huang et al., 1997; U.S. Pat. Nos. 5,877,289, 5,___,___ and5,___,___ (U.S. application Ser. Nos. 08/487,427 and 08/482,369; IssueFees paid)). A coaguligand is thus created that selectively thrombosestumor vasculature.

[0147] Truncated TF has several advantages that commend its use invascular targeted coaguligands: human tTF is readily available, and thehuman protein will have negligible or low immunogenicity in man; humantTF is fully functional in experimental animals, including mice; andtargeted tTF is highly potent because it triggers the activation of acascade of coagulation proteins, giving a greatly amplified effect (U.S.Pat. Nos. 5,877,289, 5,___,___ and 5,___,___ (U.S. application Ser. Nos.08/487,427 and 08/482,369; Issue Fees paid)).

[0148] A range of suitable target molecules that are available on tumorendothelium, but largely absent from normal endothelium, have beendescribed. For example, expressed targets may be utilized, such asendoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a TIE, a ligandreactive with LAM-1, a VEGF/VPF receptor, an FGF receptor, α_(v)β₃integrin, pleiotropin or endosialin (U.S. Pat. Nos. 5,855,866 5,877,289;Burrows et al., 1992; Burrows and Thorpe, 1993; Huang et al., 1997; Liuet al., 1997; Ohizumi et al., 1997; each incorporated herein byreference).

[0149] Adsorbed targets are another suitable group, such as VEGF, FGF,TGFβ, HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE or atumor-associated fibronectin isoform (U.S. Pat. Nos. 5,877,289,5,___,___ and 5,___,___; corresponding to U.S. Ser. Nos. 08/350,212 and08/487,427; Issue Fees paid; each incorporated herein by reference).Fibronectin isoforms are ligands that bind to the integrin family ofreceptors. Tumor-associated fibronectin isoforms are targetablecomponents of both tumor vasculature and tumor stroma. The monoclonalantibody BC-1I (Carnemolla et al., 1989) specifically binds totumor-associated libronectin isoforms.

[0150] Other targets inducible by the natural tumor environment orfollowing intervention by man are also targetable entities, as describedin U.S. Pat. Nos. 5,776,427, 5,863,538 and 5,___,___ (U.S. Ser. No.08/479,727, Issue Fee paid; each incorporated herein by reference). Whenused in conjunction with prior suppression in normal tissues and tumorvascular induction, MHC Class II antigens may also be employed astargets (U.S. Pat. Nos. 5,776,427; 5,863,538; 5,___,___ and 5,___,___(U.S. Ser. Nos. 08/295,868 and 08/479,727, Issue Fees paid); eachincorporated herein by reference).

[0151] One currently preferred target for clinical applications isvascular endothelial adhesion molecule-1 (VCAM-1) (U.S. Pat. Nos.5,855,866, 5,877,289, 5,___,___ and 5,___,___ (corresponding to U.S.Ser. Nos. 08/482,369 and 08/487,427, Issue Fees paid); each incorporatedherein by reference). VCAM-1 is a cell adhesion molecule that is inducedby inflammatory cytokines IL-1α, IL-4 (Thornhill et al., 1990) and TNFα(Munro, 1993) and whose role in vivo is to recruit leukocytes to sitesof acute inflammation (Bevilacqua, 1993).

[0152] VCAM-1 is present on vascular endothelial cells in a number ofhuman malignant tumors including neuroblastoma (Patey et al., 1996),renal carcinoma (Droz et al., 1994), non-small lung carcinoma (Staal-vanden Brekel et al., 1996), Hodgkin's disease (Patey et al., 1996), andangiosarcoma (Kuzu et al., 1993), as well as in benign tumors, such asangioma (Patey et al., 1996) and hemangioma (Kuzu et al., 1993).Constitutive expression of VCAM-I in man is confined to a few vessels inthe thyroid, thymus and kidney (Kuzu et al., 1993; Bruijn and Dinklo,1993), and in the mouse to vessels in the heart and lung (Fries et al.,1993).

[0153] Certain of the data presented herein even further supplementthose provided in U.S. Pat. No. 5,855,855 and 5,877,289 and 5,___,___(corresponding to U.S. Ser. No. 08/487,427, Issue Fee paid; eachincorporated herein by reference) and show the selective induction ofthrombosis and tumor infarction resulting from administration of ananti-VCAM-1·tTF coaguligand. The results presented were generated usingmice bearing L540 human Hodgkin lymphoma. When grown as a xenogratt inSCID mice, this tumor shows close similarity to the human disease withrespect to expression of inflammatory cytokines (Diehl et al, 1985) andthe presence of VCAM-1 and other endothelial cell activation moleculeson its vasculature.

[0154] Using a covalently-linked anti-VCAM-1·tTF coaguligand, in whichtTF was directly linked to the anti-VCAM-1 antibody, it is shown hereinthat the coaguligand localizes selectively to tumor vessels, inducesthrombosis of those vessels, causes necrosis to develop throughout thetumor and retards tumor growth in mice bearing solid L540 Hodgkintumors. Tumors generally needed to be at least about 0.3 cm in diameterto respond to the coaguligand, because VCAM-1 was absent from smallertumors. Presumably, in small tumors, the levels of cytokines secreted bytumor cells or host cells that infiltrate the tumor are too low forVCAM-1 induction. This is in accordance with the studies in U.S. Pat.Nos. 5,855,866, 5,877,289, 5,776,427, 5,___,___, and 5,___,___ (Ser.Nos. 08/487,427 and 08/479,727, Issue Fees paid), where the inventionswere shown to be most useful in larger solid tumors.

[0155] Although VCAM-1 staining was initially observed more in theperiphery of the tumor, the coaguligand evidently bound to and occludedblood transporting vessels—as it was capable of curtailing blood flow inall tumor regions. Furthermore, one of the inventors contemplates thatthe thrombin generation caused by the initial administration of thecoaguligand likely leads to further VCAM-1 induction on central vessels(Sluiter et al., 1993), resulting in an amplified signal and evidentdestruction of the intratumoral region. This type of coagulant-inducedexpression of further targetable markers, and hence signalamplification, is also disclosed in U.S. Ser. No. 08/479,727 and08/481,904 (U.S. Pat. No. 5,___,___; Issue Fee paid).

[0156] B. Mechanism of VCAM-1-Targeted Tumor Destruction

[0157] As shown herein, although localization to VCAM-1-expressingvessels in the heart and lungs of mice was observed upon administrationof an anti-VCAM-1 coaguligand, this construct did not induce thrombosisin such non-tumor sites. Furthermore, the anti-VCAM-I coaguligand was nomore toxic to mice than was a control coaguligand of irrelevantspecificity, again indicating that the constitutive expression of VCAM-1on heart and lung vessels did not lead to toxicity. This data isimportant to the immediate clinical progress of coaguligand therapy,given that VCAM-1 is a naturally occurring marker of tumor vascularendothelium in humans. However, this phenomenon also provided theinventors with a unique insight, leading to a totally different approachto tumor vasculature destruction.

[0158] The inventors sought to understand the mechanism behind theability of the anti-VCAM -1 coaguligand to bind to the VCAM-1constitutively expressed on blood vessels in the heart and lungs, andyet not to cause thrombosis in those vessels. There are numerousscientific possibilities for this empirical observation, generallyconnected with the prothrombotic nature of the tumor environment anyfibrinolytic predisposition in the heart and lungs.

[0159] Generally, there is a biological equilibrium between thecoagulation system (fibrin deposition) and the fibrinolytic system(degradation of fibrin by enzymes). However, -in malignant disease,particularly carcinomas, this equilibrium is disrupted, resulting in theabnormal activation of coagulation (hypercoagulability or the“prothrombotic state”). Evidence also indicates that various componentsof these pathways may contribute to the disorderly characteristics ofmalignancy, such as proliferation, invasion, and metastasis (Zacharskiet al., 1993).

[0160] Donati (1995) reviewed the complex interplay between the originalclinical observations of thrombotic complications of malignant diseases,and the subsequent progress in the cell biology and biochemistry oftumor cell activities. However, despite extensive research, a clearmolecular explanation for the prothrombotic nature of the tumorenvironment could not be provided (Donati, 1995). Donati did emphasize,though, the role of tumor cells in this process. It was explained thattumor cells express procoagulant activities, such as tissuethromboplastin and cancer procoagulant (CP) (Donati, 1995). WO 91/07187also reported a procoagulant activity of tumor cells.

[0161] Numerous other studies have also identified the tumor cellsthemselves as being responsible for the prothrombotic state wvitlhin atumor. For example, Nawroth et al. (1988) reported that factor(s)elaborated by sarcoma cells enhance the procoagulant response of nearbyendothelium to TNF. These authors reported that fibrin formationoccurred throughout the tumor vascular bed 30 minutes after TNFinfusion, but that fibrin deposition and platelet aggregates were notobserved in normal vasculature (Nawroth et al., 1988). TNF was latershown to enhance the expression of tissue factor on the surface ofendothelial cells (Murray et al, 1991). This was proposed to explainearlier studies showing that cultured endothelial cells incubated withrecombinant TNF have enhanced procoagulant activity, tissue factor, andconcomitant suppression of the protein C pathway, an anti-thromboticmechanism that functions on the surface of quiescent endothelial cells(Nawroth et al., 1985; Nawroth and Stem, 1986).

[0162] Data from Sugimura et al (1994) also implicated tumor cells asthe key components of the procoagulant activity of the tumor. It wasreported that four tumor cell lines were able to support differentstages of the extrinsic pathway of coagulation (Sugimura et al., 1994).Another study reported that a human ovarian carcinoma cell line,OC-2008, constitutively expressed surface membrane Tissue Factoractivity and exhibited cell surface-dependent prothrombinase complexactivity (Rao et al., 1992). Connor et al. (1989) further suggested thatit is the pathologic cells that control coagulation. Their resultsindicated that tumorigenic, undifferentiated murine erythroleukemiccells exhibit a 7- to 8-fold increase in the potency of theirprocoagulant activity (Connor et al., 1989).

[0163] Zacharski et al (1993) also focused on tumor cells and sought todefine the mode of interaction of ovarian carcinoma cells with thecoagulation (procoagulant-initiated) and fibrinolysis (urokinase-typeplasminogen activator-initiated, u-PA) pathways. They reported thattumor cells expressed Tissue Factor and coagulation pathwayintermediates that resulted in local thrombin generation—as evidenced bythe conversion of fibrinogen, present in tumor connective tissue, tofibrin that was found to hug the surfaces of tumor nodules andindividual tumor cells. Detected fibrin could not be accounted for onthe basis of necrosis or a local inflammatory cell infiltrate (Zacharskiet al., 1993). These authors concluded that there exists a dominanttumor cell-associated procoagulant pathway that leads to thrombingeneration and hypercoagulability.

[0164] Other hypotheses have proposed that it is changes in the tumorblood vessels that render these vessels better able to support theformation of thrombi and/or less able to dissolve fibrin. For example,tumor vessels have been reported to exhibit upregulation of TissueFactor, down-regulation of plasminogen activators and/or upregulation ofthe inhibitor of plasminogen activators, PAI-1 (Nawroth and Stern, 1986;Nawroth et al., 1988). Such effects are believed to be magnified bytumor derived factors (Murray et al., 1991; Ogawa et al., 1990),possibly VEGF.

[0165] For example, Ogawa et al. (1990) reported that hypoxia causedendothelial cell surface coagulant properties to be shifted to promoteactivation of coagulation. This was accompanied by suppression of theanticoagulant cofactor, thrombomodulin, and induction of an activator offactor X, distinct from the classical extrinsic and intrinsic systems(Ogawa et al., 1990). Also, there could be an increase in the localconcentration of Factors VIIa, IXa, Xa, or other molecules that interactwith TF, within the tumor vessels, thus encouraging thrombosis.

[0166] Additionally, platelets are a major component of any procoagulantstate. Recently, the procoagulant potential of platelets has been linkedto their ability to shed procoagulant microparticles from the plasmamembrane (Zwaal et al., 1989; 1992; Dachary-Prigent et al., 1996). Ithas been proposed that an increased proportion of circulatingmicroparticles, vesicles or membrane fragments from plateletscontributes to ′prethrombotic′ (prothrombotic) states in variouspathological conditions (Zwaal et al., 1989; 1992; Dachary-Prigent etal., 1996, pp. 159 and references cited therein). McNeil et al. (1990)also reported that β₂-GPI exerts multiple inhibitory effects oncoagulation and platelet aggregation. Tumor platelet biology could thusexplain the effectiveness of the anti-VCAM-1 coaguligand.

[0167] Further tenable explanations included the simple possibility thatVCAM-1 is expressed at higher levels in tumor vessels than on bloodvessels in the heart and lungs, probably due to induction bytullmor-derived cytokines, and that binding to the healthy vesselscannot tip the balance into sustained thrombosis. Also the fibrinolyticmechanisms could be upregulated in the heart, as exemplified byincreased Tissue Factor pathway inhibitor (TFPI), increased plasminogenactivators, and/or decreased plasminogen activator inhibitors. Shouldthe fibrinolytic physiology of the heart and lung vessels prove to bethe major reason underlying the tumor-specific effects of theanti-VCAM-1 coaguligand, this would generally preclude the developmentof additional anti-tumor therapies targeted to unique aspects of tumorbiology.

[0168] Despite all the possible options, the inventors reasoned that thefailure of the anti-VCAM -1 coaguligand to cause thrombosis in vesselsof normal tissues was due to the absence of the aminophospholipid,phosphatidylserine (PS), from the luminal surface of such vessels. Tocomplete the theory, therefore, not only would phosphatidylserine haveto be shown to be absent from these normal vessels, but its presence onthe luminal side of tumor-associated vessels would have to beconclusively demonstrated.

[0169] The inventors therefore used immunohistochemical staining toevaluate the distribution of a monoclonal anti-phosphatidylserine(anti-PS) antibody injected intravenously into tumor-bearing mice. Thesestudies revealed that the VCAM-1 expressing vessels in the heart andlungs lacked PS, whereas the VCAM-1 expressing vessels in the tumorexpressed PS. The need for surface PS expression in coaguligand actionis further indicated by the inventors' finding that annexin V, whichbinds to PS, blocks anti-VCAM-1·tTF coaguligand action, both in vitroand in vivo The lack of thrombotic effect of the anti-VCAM-1 coaguligandon normal heart and lung vessels can thus be explained, at least inpart: the absence of the aminophospholipid, phosphatidylserine, meansthat the normal vessels lack a procoagulant surface upon whichcoagulation complexes can assemble. In the absence of surface PS,anti-VCAM-1 ·tTF binds to VCAM-1 expressing heart and lung vessels, butcannot induce thrombosis. In contrast, VCAM-1 expressing vessels in thetumor show coincident expression of surface PS. The coaguligand thusbinds to tumor vessels and activates coagulation factors locally to forman occlusive thrombus.

[0170] In addition to delineating the tumor-specific thrombotic effectsof anti-VCAM-I coaguligands, the specific expression of theaminophospholipid, phosphatidylserine, on the luminal surface of tumorblood vessels also allowed the inventors to explain the prothromboticphenotype observed, but not understood, in earlier studies (Zacharski etal., 1993; Donati, 1995). Rather than being predominantly due to tumorcells or elaborated factors; platelets, procoagulant microparticles ormembrane fragments; or due to imbalances in thromboplastin,thrombomodulin, cancer procoagulant, Tissue Factor, protein C pathway,plasminogen activators or plasminogen activator inhibitors (e.g.,PAI-1), the inventors' studies indicate that it is PS expression thatplays a significant role in the prothrombotic state of tumorvasculature.

[0171] C. Aminophospholipids as Markers of Tumor Vasculature

[0172] Following their discovery that the representativeaminophospholipid, phosphatidylserine, was specifically expressed on theluminal surface of tumor blood vessels, but not in normal blood vessels,the inventors reasoned that aminophospholipids had potential as targetsfor therapeutic intervention. The present invention thereforeencompasses targeting the aminophospholipid constituents, particularlyphosphatidylserine (PS) and phosphatidylethanolamine (PE), in tumortreatment. Although anti-tumor effects from aminophospholipid targetingare demonstrated herein, using art-accepted animal models, the abilityof aminophospholipids to act as safe and effective targetable markers oftumor vasculature could not have been predicted from previous studies.

[0173] For example, although tumor vessels are generally prothromboticin nature, as opposed to other blood vessels, it is an inherent propertyof the tumor to maintain a network of blood vessels in order to deliveroxygen and nutrients to the tumor cells. Evidently, tumor-associatedblood vessels cannot be so predisposed towards thrombosis that theyspontaneously and readily support coagulation, as such coagulation wouldnecessarily cause the tumor to self-destruct. It is thus unexpected thatany thrombosis-associated tumor vessel marker, such as the presentlyidentified phosphatidylserinie, could be discovered that is expressed inquantities sufficient to allow effective therapeutic intervention bytargeting, and yet is expressed at levels low enough to ordinarilymaintain blood flow through the tumor.

[0174] The present identification of aminophospholipids as safe andeffective tumor vasculature targets is even more surprising given (I)the previous speculations regarding the role of other cell types and/orvarious factors, activators and inhibitors underlying the complex,prothrombotic state of the tumor (as discussed above); and (2) theconfusing and contradictory state of the art concerningaminophospholipid biology, in terms of both expression and function invarious cell types.

[0175] Phosphatidylserine and phosphatidylethanolamine are normallysegregated to the inner surface of the plasma membrane bilayer indifferent cells (Gaffet et al., 1995; Julien et al., 1995). In contrast,the outer leaflet of the bilayer membrane is rich in phosphatidylcholineanalogs (Zwaal et al., 1989; Gaffet et al., 1995). This lipidsegregation creates an asymmetric transbilayer. Although the existenceof membrane asymmetry has been discussed for some time, the reason forits existence and the mechanisms for its generation and control arepoorly understood (Williamson and Schlegel, 1994), particularly in cellsother than platelets.

[0176] There are even numerous conflicting reports regarding thepresence or absence of PS and PE in different cells and tissues, letalone concerning the likely role that these aminophospholipids may play.For example, the many PS studies conducted with platelets, keycomponents in blood coagulation (Dachary-Prigent et al., 1996), haveyielded highly variable results. Bevers et al. (1982) measured theplatelet prothrombin-converting activity of non-activated plateletsafter treatment with various phospholipases or proteolytic enzymes. Theyconcluded that negatively charged phosphatidylserine, and possiblyphosphatidylinositol, were involved in the prothrombin-convertingactivity of non-activated platelets (Bevers et al., 1982).

[0177] Bevers et al. (1983) then reported an increased exposure ofphosphatidylserine, and a decreased exposure of sphingomyelinase, inactivated platelets. However, these alterations were much less apparentin platelets activated either by thrombin or by collagen alone, incontrast to collagen plus thrombin, diamide, or a calcium ionophore(Bevers et al., 1983). The surface expression of PS in response todiamide was contradicted by studies in erythrocytes, which showed nodiamide-stimulated PS exposure (de Jong et al., 1997). While echoingtheir earlier results, Bevers and colleagues then later reported thatchanges in the plasma membrane-cytoskeleton interaction, particularlyincreased degradation of cytoskelet al actin-binding protein, wasimportant to platelet surface changes (Bevers et al., 1985; pages368-369).

[0178] Maneta-Peyret et al. (1989) also reported the detection of PS onhuman platelets. These authors noted that the platelet procoagulantsurface could be formed by negatively charged phospholipids, such asphosphatidylserine and phosphatidylethanolamine (generally neutral orzwitterionic), or both. The role of phosphatidylserine in the process ofcoagulation has been questioned in favor of phosphatidylethanolamine(Maneta-Peyret et al., 1989; Schick et al., 1976; 1978). For example,studies have reported that 18% of phosphatidylethanolamine becomessurface-accessible after 2 hours, in contrast to zero phosphatidylserine(Schick et al., 1976).

[0179] Ongoing studies with platelets were also reported as showing afurther 16% increase in phosphatidylethanolamine exposure after thrombintreatment, with no increase in the phosphatidylserine levels (Schick etal., 1976). Therefore, PS was said not to be a component of thefunctional surface of the platelet plasma membrane (Schick et al., 1976;1978). Nonetheless, current evidence does seem to indicate that both PSand PE are involved in the phospholipid asymmetry observed in the outermembrane of platelets and erythrocytes, and that PS is involved in theprocoagulant activity of platelets (Gaffet et al., 1995; de Jong et al.,1997; U.S. Pat. No. 5,627,036).

[0180] The mechanisms for achieving and maintaining differentialaminophospholipid distribution, let alone the functional significance ofdoing so, have long been the subject of controversial speculations. Inreviewing the regulation of transbilayer phosplholipid movement,Williamson and Schlegel (1994) indicated that elevating intracellularCa2′ allows the major classes of phosplholipids to move freely acrossthe bilayer, scrambling lipids and dissipating asymmetry. de Jong et al.(1997) also reported that an increase of intracellular calcium leads toa rapid scrambling of the lipid bilayer and the exposure of PS. whichcould be partially inhibited by cellular oxidation. The interaction ofaminophospholipids with cytoskelet al proteins has also been proposed asa mechanism for regulating membrane phospholipid asymmetry (Zwaal etal., 1989).

[0181] Gaffet et al. (1995) stated that the transverse redistribution ofphospholipids during human platelet activation is achieved by avectorial outflux of aminophospholipids, not counterbalanced by a rapidreciprocal influx of choline head phospholipids, i.e. not scrambling.They suggested that the specific vectorial outflux of aminophospholipidscould be catalyzed by a “reverse aminophospholipid translocase” activity(Gaffet et al., 1995). An alternative hypothesis would be that theactivity of an inward translocase was inhibited. Zwaal et al. (1989)proposed the involvement of a phospholipid-translocase that catalyzedboth the outward and inward movement of aminophospholipids.

[0182] The presence of an energy- and protein-dependentaminophospholipid translocase activity that transportsphosphatidylethanolamine from the outer to the inner leaflet of thelipid bilayer was reported by Julien et al. (1993). They then showedthat the aminophospholipid translocase activity could also transferphosphatidylserine, and that the activity could be maintained,suppressed and restored depending on the conditions of cell incubation(Julien et al., 1993), and inhibited by the tumor promoter,12-O-tetradecanoylphorbol-13-acetate (TPA) (Julien et al., 1997).

[0183] A 35 kD phospholipid scramblase that promotes the Ca²⁺-dependentbidirectional movement of phosphatidylserine and other phospholipids wasrecently cloned from a CDNA library (Zhou et al., 1997). This “PLscramblase” protein is a proline-rich, type II plasma membrane proteinwith a single transmembrane segment near the C terminus. Subsequentstudies confirmed that this protein was responsible for the rapidmovement of phospholipids from the inner to the outer plasma membraneleaflets in cells exposed to elevated cytosolic calcium concentrations(Zhao et al, 1998).

[0184] The aminophospholipid translocase activity reported by Julien etal. (1993; 1997), which transports PS and PE from the outer to the innerleaflet, is different to the bidirectional Ca²⁺-dependent scramblase(Zhou et al., 1997; Zhao et al., 1998). The scramblase is activated byCa²⁺, and mostly functions to move PS from the inner to the outerleaflet in response to increased Ca²⁺ levels. It is now generallybelieved that the aminophospholipid translocase maintains membraneasymmetry during normal conditions, but that the scramblase is activatedby Ca²⁺ influx, over-riding the translocase and randomizingaminophospholipid distribution.

[0185] The normal segregation of PS and PE to the inner surface of theplasma membrane is thus now generally accepted, and certain membranecomponents involved in the asynmmetric processes have even beenidentified. However, doubts remain about the conditions, mechanisms andcell types that are capable of re-locating aminophospholipids to theouter leaflet of the membrane, and the biological implications of suchevents.

[0186] Contradictory reports concerning aminophospholipid expression arenot limited to studies of platelets. Phosphatidylserine andphosphatidylethanolarnine are generally about 7% and about 10%,respectively, of the phospholipid composition of cultured humanendothelial cells from human artery, saphenous and umbilical vein (7.1%and 10.2%, respectively; Murphy et al., 1992). However, an importantexample of the contradictions in the literature concerns the ability ofanti-PS antibodies to bind to endothelial cells (Lin et al, 1995).

[0187] The anti-PS antibodies present in recurrent pregnancy loss (Roteet al., 1995; Rote, 1996; Vogt et al., 1996; Vogt et al., 1997) werebelieved to modulate endothelial cell function, without evidence ofbinding to endothelial cells. In an attempt to explain this discrepancy,Lin et al. (1995) tried but failed to demonstrate anti-PS antibodybinding to resting endothelial cells. They concluded that PS antigenicdeterminants are not expressed on the surface of resting endothelialcells, although a PS-dependent antigenic determinant was associated withcytoskclet al-like components in acetone-fixed cells (Lin et al, 1995).

[0188] Van Heerde et al. (1994) reported that vascular endothelial cellsin vitro can catalyze the formation of thrombin by the expression ofbinding sites at which procoagulant complexes can assemble. In contrastto other studies with activated platelets (Bevers et al., 1982; 1983;1985; Maneta-Peyret et al., 1989; Schick et al, 1976; 1978), stimulatedHUVEC endothelial cells did not exhibit an increase in PS binding sitesas compared to quiescent cells (Van Heerde el al., 1994).Phosphatidylserine was reported to be necessary for Factor Xa formationvia the extrinsic as well as the intrinsic route (Van Heerde et al.,1994). Nonetheless, Brinkman et al. (1994) published contradictoryresults, indicating that other membrane constituents besides negativelycharged phospholipids are involved in endothelial cell mediated,intrinsic activation of factor X.

[0189] Ravanat et al. (1992) also studied the catalytic potential ofphospholipids in pro- and anti-coagulant reactions in purified systemsand at the surface of endothelial cells in culture after stimulation.Their seemingly contradictory results were proposed to confirm a rolefor phospholipid-dependent mechanisms in both procoagulant Tissue-Factoractivity and anticoagulant activities (activation of protein C by thethrombin-thrombomodulin complex and by Factor Xa) (Ravanat et al.,1992). The Ravanat et al. (1992) results were also said to provideevidence of phospholipid exposure during activation of human endothelialcells, which was not observed by Van Heerde et al. (1994) or Brinkman etal. (1994). However, they did note that anionic phospholipids are ofrestricted accessibility in the vicinity of cellular Tissue Factor. Thesituation is further complicated as, even after Tissue Factor induction,other events are likely necessary for coagulation, as the Tissue Factorremains inaccessible, being under the cell.

[0190] Ravanat et al. (1992) went on to suggest that the differentextent of inhibition of Tissue Factor and thrombomodulin activities onstimulated endothelial cells means that the cofactor environments differfor the optimal expression of these opposite cellular activities.However, the acknowledged difficulties in trying to reproduce exactcellular phospholipid environments (Ravanat et al., 1992), raise thepossibility of artifactual data from these in vitro studies. Indeed,irrespective of the Ravanat el al. (1992) data, it is generallyacknowledged that meaningful information regarding tumor biology, andparticularly therapeutic intervention, can only be gleaned from in vivostudies in tumor-bearing animals, such as those conducted by the presentinventors.

[0191] In addition to the disagreements regarding aminophospholipidexpression, as discussed above, there are also conflicting reportsconcerning the function of aminophospholipids in various cell types.Although it is now generally accepted that PS expression on activatedplatelets is connected with the procoagulant surface, in discussing thephysiological significance of membrane phospholipid asymmetry inplatelets and red blood cells, Zwaal et al. (1989) highlighted otherimportant functions. Moreover, Toti et al. (1996) stated that thephysiological implications of a loss of asymmetric phospholipiddistribution remain poorly understood in cell types other than bloodcells.

[0192] Zwaal et al. (1989) stated that the membrane phospholipidasynmmetry of platelets and red cells is undone when the cells areactivated in various ways, presumably mediated by the increasedtransbilayer movement of phospholipids. These changes, coupled with therelease of shed microparticles, were explained to play a role in localblood clotting reactions. A similar phenomenon was described to occur insickled red cells: phospholipid vesicles breaking off from reversiblysickled cells contribute to intravascular clotting in the crisis phaseof sickle cell disease (Zwaal et al., 1989).

[0193] Both Zwaal et al. (1989) and Williamson and Schlegel (1994) haveindicated that the physiological significance of surface phospholipidchanges is not restricted to hemostasis. In fact, the surface exposureof PS by blood cells was said to significantly alter their recognitionby the reticuloendothelial system, and was to likely represent at leastpart of the homeostatic mechanism for the clearance of blood cells fromthe circulation (Zwaal et al., 1989). Thus, PS acts as a signal for theelimination of activated platelets after bleeding has stopped.Recognition of PS exposed on sickle cells and malarially infected cellsby phagocytes and macrophages explains their counter-pathophysiologicaleffects (Zwaal et al., 1989). Furthermore, PS-dependent phagocytosismarks virally infected cells for phagocytic uptake (WO 97/17084). Thesurface expression of arninophospholipids could also confer “fusioncompetence” to a cell (Williamson and Schlegel, 1994).

[0194] Williamson and Schlegel (1994) also speculated that there is amore general raivon d′etre for lipid asymmetry. For example, althoughthe different head groups have received most attention, it could well bethat fatty acid asymmetry is the important factor (Williamson andSchlegel, 1994). A further hypothesis is that the asymmetricdistribution of transbilayer phospholipids has no function in itself,but that it is the dynamic process of lipid movement that is importantto biological systems (Williamson and Schlegel, 1994).

[0195] Many groups have reported that turnor cells are responsible forthe prothrombinase activity of the tumor (Connor et al., 1989; Rao etal., 1992; Zacharski et al., 1993; Sugimura et al., 1994; Donati, 1995).This could have been reasoned to be due to PS (WO 91/07187). However,the results of Sugimura et al. (1994) argue against this: they reportedthat although both the prothrombinase activity and total procoagulantactivity of the tumorigenic cells, HepG2 and MKN-28, fell on reachingconfluency, the PS levels remained constant.

[0196] Rather than supporting a role for tumor cell PS inprothirombinase activity, Connor et al. (1989) suggested that theincreased expression of PS in tumorigenic cells is relevant to theirability to be recognized and bound by macrophages. Utsugi et al. (1991)similarly proposed that the presence of PS in the outer membrane ofhuman tumor cells explains their recognition by monocytes.

[0197] Jamasbi et al. (1994) suggested a totally different role forlipid components in tumorigenic cells, proposing that the lipidsinterfere with tumor antigen accessibility. Thus, tumor cell lipidswould act to modify the tumor cell surface antigen(s), thus protectingthe tumor cells from host immune destruction (Jarnasbi et al., 1994).This hypothesis is not unlike that proposed by Qu et al. (I1996), interms of endothelial cells. These authors showed that T cells adhered tothrombin-treated human umbilical endothelial cells by virtue ol bindingto PS (Qu el al, 1996).

[0198] It has thus been proposed that PS-mediated T cell adhesion toendothelial cells in vivo is important to both immune surveillance, andalso to the disease processes of atherosclerosis (Qu et al., 1996;Moldovan et al., 1994). Bombeli et al. (1997) and Flynn et al. (1997)also suggested that cells within atherosclerotic plaques may contributeto disease progression by exposing PS, although this was based solely onin vitro studies. Qu et al. (1996) and Moldovan et al. (1994) evenhinted at an approach opposite to that of the present invention, i.e.,the manipulation of phosphatidylserine interactions as an anticoagulantapproach. U.S. Pat. Nos. 5,658,877 and No. 5,296,467 have proposedannexin (or “annexine”) for use as anti-endotoxins and anti-coagulants.U.S. Pat. No. 5,632,986 (incorporated herein by reference) suggests theuse of the phosphatidylserine-binding ligand, annexin V, as a conjugatewith a component, such as urokinase, that lyses thrombi.

[0199] Toti el al. (1996) suggested that Scott syndrome, an inheritedbleeding disorder, may reflect the deletion or mutation of a putativeoutward phosphatidylserine translocase or “scramblase”. Although aninteresting notion, Stout et al. (1997) later isolated a membraneprotein from Scott erythrocytes that exhibited normal PL scramblaseactivity when reconstituted in vesicles with exogenous PLs. It wassuggested that the defect in Scott syndrome is related to an alteredinteraction of Ca²⁺ with PL scramblase on the endofacial surface of thecell membrane, due either to an intrinsic constraint upon the protein,preventing interaction with Ca²+in situ, or due to an unidentifiedinhibitor or cofactor in the Scott cell that is dissociated by detergent(Stout et al., 1997).

[0200] More variable results have been reported in connection with thepossible role of PS in apoptosis. Williamson and Schlegel (1994)discussed the theme of PS as a marker of programmed cell death (PCD orapoptosis). It is generally accepted that programmed cell death, atleast in the hematopoietic system, requires the phagocytic sequestrationof the apoptopic cells before the loss of membrane integrity or“rupture”. The loss of membrane asymmetry in apoptopic cells, andparticularly the appearance of PS in the external leaflet, was proposedto be the trigger for their recognition by phagocytic macrophages(Williamson and Schlegel, 1994).

[0201] Martin el al. (1995) further reported PS externalization to be anearly and widespread event during apoptosis of a variety of murine andhuman cell types, regardless of the initiating stimulus. They alsoindicated that, under conditions in which the morphological features ofapoptosis were prevented (macromolecular synthesis inhibition,overexpression of Bcl-2 or Abi), the appearance of PS on the externalleaflet of the plasma membrane was similarly prevented (Martin et al.,1995).

[0202] However, other analyses argue against the Williamson and Schlegel(1994) and Martin et al. (1995) proposals to some extent (Vermes et al.,1995). Although these authors indicate that the translocation of PS tothe outer membrane surface is a marker of apoptosis, they reason thatthis is not unique to apoptosis, but also occurs during cell necrosis.The difference between these two forms of cell death is that during theinitial stages of apoptosis the cell membrane remains intact, while atthe very moment that necrosis occurs the cell membrane loses itsintegrity and becomes leaky. Therefore, according to this reasoning, PSexpression at the cell surface does not indicate apoptosis unless a dyeexclusion assay has been conducted to establish cell membrane integrity(Vermes et al., 1995).

[0203] Nonetheless, the body of literature prior to the presentinvention does seem to indicate that the appearance of PS on the outersurface of a cell identifies an apoptotic cell and signals that cell'singestion (Hampton et al., 1996; WO 95/27903). Hampton et al. (1996)concluded that while an elevation of intracellular Ca²⁺ was anineffective trigger of apoptosis in the cells investigated,extracellular Ca²⁺ was required for efficient PS exposure duringapoptosis. In contrast, the proposal of Martin et al. (1995) thatactivation of an inside-outside PS translocase is an early widespreadevent during apoptosis would seem to require at least some intracellularCa²⁺ (Zhou et al., 1997; Zhao et al., 1998).

[0204] Blankenberg el al. (1998) very recently reported that annexini V,an endogenous human protein with a high aftinity for PS, can be used toconcentrate at sites of apoptotic cell death in vivo. Radiolabeledannexin V localized to sites of apoptosis in three models, includingacute cardiac allograft rejection (Blankenberg et al., 1998). Stainingof cardiac allografts for exogenously administered annexin V revealedmyocytes at the periphery of mononuclear infiltrates, of which only afew demonstrated positive apoptotic nuclei.

[0205] Finally, the transbilayer movement of phospholipids in the plasmamembrane has even been analyzed in ram sperm cells, where the existenceof a transverse segregation of phospholipids has been implicated in thefertilization process (Muiller et al., 1994). Phospholipid asymmetry hasthus been receiving increasing attention, although a clear understandingof this phenomenon, or its relationship to health or disease, has notbeen realized.

[0206] Irrespective of the confuising state of the art regardingaminophospholipid biology, the present inventors discovered, incontrolled in vivo studies, that aminophospholipids, such as PS and PE,were specific markers of tumor blood vessels. This is surprising inlight of the earlier studies of aminophospholipid fimction, particularlythose indicating that the cell surface expression of PS is accompaniedby binding of circulating cells, such as T cells (Qu et al., 1996),macrophages (Connor et al., 1989), monocytes (Utsugi et al., 1991) orphagocytes (Zwaal et al., 1989; Williamson and Schlegel, 1994) and is amarker of apoptopic cells (Hampton et al., 1996; Martin et al., 1995;Zhou et al., 1997; Zhao et al., 1998).

[0207] Thus, prior to this invention, the possibility of usingaminophospholipids as targetable markers of any disease, let alone oftumor vasculature, would be unlikely to be contemplated, due to theperceived masking of these molecules by the binding of one or more celltypes, or their transient expression before apoptopic death. In fact,speculative suggestions have concerned the disruption of PS-cellularinteractions, such as in preventing leukocyte binding, an initial eventin atherosclerosis (Qu et al., 1996).

[0208] Other surprising aspects of this discovery are evident in acomparison to earlier work concerning the shedding of procoagulantmicroparticles from plasma membranes and the demarcation of cells forphagocytosis (WO 97/17084). Zwaal el CIL (1989; 1992) andDachary-Prigent et al. (1996) explained that PS translocation to theplasma membrane is followed by release of microparticles, microvesiclesor microspheres from the cells. Zwaal et al. (1989) and Williamson andSchlegel (1994) indicated that PS surface expression prompts clearanceby the reticuloendothelial system. In light of these fates ofPS-expressing cells, and the various documented bilayer translocaseactivities (Julien et cia, 1995; Zhou et al., 1997; Zhao el al., 1998),it is surprising that cell surface aminophospholipids such as PS and PEcan form static and stable enough markers to allow antibody localizationand binding.

[0209] Prior to the present invention, there was mounting evidence thatsurface PS appears as ′ part of the apoptopic process, marking cells forrapid destruction (Hampton et al., 1996; Martin et al., 1995).Therefore, although reasonable for use as a diagnostic marker forcertain disease states, such as graft rejection (Blankenberg et al.,1998), the apparently limited life time of surface PS would also adviseagainst its use as a viable marker for targeting in therapeuticintervention.

[0210] Nonetheless, the present study did indeed discoveraminophospholipids to be markers of tumor vascular endothelial cellssuitable for targeting. After postulating that PS expression wasnecessary for VCAM coaguligand action, the presence of PS on tumor bloodvessels, but normal vessels, was demonstrated in vivo. The in vivoobservations allowed the inventors to explain the safety andeffectiveness of the anti-VCAM coaguligands. This is due to therequirement for coincident expression of a targeted marker (e.g., VCAM)and PS on tumor endothelium. Even if the target molecule is present onendothelium in normal or pathological conditions, thrombosis will notresult if surface PS expression is lacking.

[0211] The value of the present invention is not limited to explainingcoaguligand action, nor to the surprising development of naked antibodytherapies. These discoveries have allowed the inventors to show, for thefirst time, that PS translocation in endothelial cells can occur withoutsignificant cell damage or cell death (Example XIV). In the inventors'new model of tumor biology, the translocation of PS to the surface oftumor blood vessel endothelial cells occurs, at least in a significantpart. independently of apoptopic or other cell-death mechanisms. Thus,PS surface expression in the tumor environment is not a consequence ofcell death, nor does it trigger immediate cell destruction. This is offundamental importance and represents a breakthrough in the scientificunderstanding of PS biology, membrane translocation, cell signaling andapoptosis pathways.

[0212] The separation of endothelial cell PS translocation fromapoptosis (Example XIV) is also integral to methods of therapeuticintervention based upon PS surface expression. Should PS translocationto the outer membrane in tumor vascular endothelial cells occur only indying cells, or should it inevitably trigger cell death, then the PSmarker would not likely be sufficiently available to serve as atargetable entity for successful therapy (using either naked antibodiesor therapeutic conjugates). That is not to say that PS expression oncertain tumor vascular endothelial cells is not transient, and thatturnover and cell death do not occur in this endothelial cellpopulation, but the finding that significant stable PS expression can beachieved without cell death is a landmark discovery important to variousfields of biology and to new therapies.

[0213] D. Naked Antibodies Against Aminophospholipids for TumorTreatment

[0214] The present aminophospholipid tumor vasculature expressionstudies further support the use of coaguligands directed against knowntumor vasculature markers as selective thrombotic agents for thetreatment of solid tumors. The present observations also led theinventors to develop further tumor treatment methods. For example, theuse of anti-aminophospholipid immunotoxins and anti-aminophospholipidcoaguligands in tumor treatment is disclosed and claimed in first andsecond provisional applications Ser. Nos. 60/092,589 (filed Jul. 13,1998) and 60/110,600 (filed Dec. 02, 1998) and in co-filed U.S. and PCTpatent applications (Attorney Docket Nos. 4001.002300, 4001.002382,4001.002383 and 4001.002210), each specifically incorporated herein byreference. The surprising discovery of stable PS expression on intacttumor-associated endothelial cells, which are not undergoing cell death,renders such methods both practicable and surprising (given that PSexpression was thOughIl to be associated only with cell destruction).

[0215] However, yet even more unexpected methods of tumor treatment werethen discovered. In investigating the potential use of aminophospholipidtargeting, in the context of later delivering a toxin or coagulant tothe tumor vasculature, the inventors serendipitously discovered thatnaked anti-PS antibodies have a destructive effect on tumor vasculaturein vivo—in the complete absence of any additional effector moiety.

[0216] One of the present inventors has been developing anti-tumorvasculature immunotoxins and coaguligands for therapeutic use for sometime (for example, see U.S. Pat. Nos. 5,5855,866 and 5,877,289; and U.S.application Ser. Nos. 08/350,212, 08/487,427 and 08/482,369; Issue Feespaid; each incorporated herein by reference). In the normal course ofthese studies, various antibodies, including anti-Class II,anti-endoglin, anti-VCAM-1 and anti-VEGF, have been administered totumor-bearing animals and shown to specifically localize to theintratumoral vasculature. Following such confirmation, the antibodiesare linked to the toxic or coagulative effector portion to form animmunotoxin or coaguligand, which is then administered to exert ananti-tumor effect.

[0217] During such studies, no naked antibodies have been found to exertan anti-tumor effect in themselves. The ability ofanti-aminophospholipid antibodies to both specifically localize to tumorvasculature and to exert a concomitant destructive effect, leading totumor necrosis, is therefore most unexpected.

[0218] Although a precise molecular understanding of exactly how thenaked antibodies function is not necessary in order to practice thepresent invention, the inventors have contemplated several mechanismsthat may account for the observed endothelial cell killing. The favoredmechanisms include cell-mediated cytotoxicity, complement-mediated lysisand/or apoptosis, although antibody-induced cell signaling and/ordisturbances to the cytoskeleton may also be involved.

[0219] As the naked or unconjugated anti-aminophospholipid antibodies orantibody fragments bind to aninophospholipids at the surface of thetumor vascular endothelial cells, they will form an antibody coating onthe luminal surface. This may function to attract immune eflcctor cells,such as cytotoxic Tr cells and/or NK cells, which will then exert acell-mediated cytotoxic effect on the vascular endothelial cells.

[0220] Binding of intact anti-aminophospholipid antibodies to thevascular endothelial cell surface will also mean that the Fe portions ofthe antibodies will protrude into the vessel lumen. As antibody Fefragments activate the complement pathway, the observed cellulardestruction may be a result of complement-directed lysis. Antibodybinding thus activates the complement-dependent coagulation cascade,causing multi-component complexes to assemble and, ultimately, togenerate a lytic complex that permeabilizes the target cell.“Complement-activated ADCC” may also be operating in the destruction, inwhich complement binds to the antibody-coated target cell, and in whichcells, such as neutrophils, having receptors for complement, lyse thetarget cell.

[0221] Anti-aminophospholipid antibody binding may also induce apoptosisin the tumor vascular endothelial cells. Other groups have identified PSas a possible marker of apoptosis (Williamson and Schlegel, 1994).However, these previous studies were connected with the appearance ofexternalized PS after other stimuli had initiated the apoptopic event(Martin et al., 1995), the inverse of the present apoptosis inductionproposal. There are no known reports of antibody binding to PS actuallyinducing apoptosis. Still, the inventors consider this to be about aslikely a mechanism as the cell-mediated cytotoxicity orcomplement-mediated lysis, despite the fact that tangential evidence tothe contrary has very recently been published by others (Nakamura etal., 1998).

[0222] Nakamura et al (1998) analyzed antibody fractions from patientswith lupus anticoagulant (LAC), a disorder associated with arterial andvenous thrombosis, thrombocytopenia, and recurrent fet al loss. Plasmawith LAC activity was initially reported to induce apoptosis inendothclial cells (Nakamura el al., 1994). The apoptotic activities ofLAC antisera were then reported to be localized in an annexin V-bindingantibody fraction in 10/10 patients studied (Nakamura el al., 1998). Asannexin binds to PS, the apparent ability of anti-annexin antibodies toinduce apoptosis would be the opposite of one of the destructivemechanisms proposed by the present inventors, i.e., the ability ofanti-PS antibodies to induce apoptosis.

[0223] The ability of LAC antibody fractions to induce apoptosis wasfurther reported to be inhibited by preincubation with annexin V(Nakamura et al., 1998). In contrast, removal of anti-phospholipidantibodies from the patients' IgG fractions with phospholipid liposomesdid not abolish the apoptosis-inducing activities or annexin V binding(Nakamura et al., 1998). These results reasonably implied that patientswith LAC often have antibodies that do not bind phospholipids and yetare responsible for the induction of apoptosis in endothelial cells(Nakamura et al., 1998).

[0224] Without needing to equate the Nakamura et al. (1998) LAC datawith the present observations from in vivo studies of tumors and tumorvasculature, due to the evidently disparate nature of these clinicalconditions, the present inventors nonetheless have certain unifyingtheories. Nakamura et al. (1998) attempted to remove anti-phospholipidantibodies from patients' antisera using phospholipid liposomes, andobserved that this did not abolish the apoptosis-inducing activity.These results led Nakamura et al. (1998) to conclude that theanti-phospholipids antibodies cannot be responsible for apoptopicactivity. However, the present inventors now have the insight to suggestthat the incubation with phospholipid liposomes may not have removed theanti-phospholipids antibodies from the antisera, as phospholipids areantigenically neutral in bilayer and liposomal form, and largely onlybind antibodies in hexagonal form (Rauch et al., 1986; Rauch and Janoff,1990; Berard et al, 1993; each incorporated herein by reference) or inassociation with membrane proteins. Thus, anti-phospholipids antibodiesmay remain in the LAC antisera and may cause, or contribute to, theobserved apoptopic activity.

[0225] It is also conceivable that anti-aminophosplholipid binding tothe surface of tumor vascular endothelial cells may cause disturbancesin the cytoskelet alal organization of the cell. As the cytoskeletonplays a role in the organization of surface membranes, and asanti-aminophospholipid binding may disturb (or further disturb) themembrane, antibody binding may transmit changes to cytoskelet alproteins that interact with the bilayer. It is already known that thespatial organization of cytoskelet al proteins controls membranestability and cell shape, and it is possible that perturbation of somecytoskelet al equilibrium may have far-reaching consequences on cellintegrity.

[0226] A further mechanism of operation of the invention may be thatanti-aminophospholipid antibody binding to the endothelial cell surfacemay initiate signal transduction by, as yet, undefined pathways.Anti-aminophospholipid antibody binding may also disturb known signaltransduction pathways, e.g., by altering the conformation and/orinteractions of membrane receptors, signal transduction proteins,membrane channels, and the like. Signals for cell destruction(apoptosis) may be initiated or mimicked, and/orpreservation/homeostatic signals may be inhibited.

[0227] Although of scientific interest, determining the exact nature ofthe vascular destruction achieved by the naked anti-aminophospholipidantibodies is not necessary to the practice of the present invention.Given that the administration of anti-aminophospholipid antibodies isherein shown to advantageously result in specific anti-tumor effects invivo, the invention can be utilized irrespective of the molecularmechanism that underlies this phenomenon.

[0228] The naked antibody use of the present invention thus represent asignificant advance in tumor therapy. Although coaguligands areadvantageous for tumor therapy, the targeting antibody or ligand stillneeds to be conjugated to, or functionally associated with, the effectorcoagulant, such as Tissue Factor. Therefore, the practice of thecoaguligand targeting and tumor destruction methodology is somewhatlaborious in that it requires the preparation of suitable conjugates, orco-ordinated molecular complexes (including bispecific antibodies). Forexample, one must prepare a targeting antibody or ligand that binds tothe desired target antigen; choose an appropriate coagulant; link thecoagulant to the targeting antibody or ligand, or otherwise form afunctional association of the two components, to form the “coaguligand”;separate the coaguligand from the unconjugated, or unlcomplcxed,targeting agent and coagulant; and then conduct the treatment protocols.

[0229] Although coaguligand-based methods can be readily andsuccessfully practiced, one can see the advantages that result from thepresent development of methodology that includes less preparative stepsand can therefore be performed in a more cost-effective manner.Furthermore, the present invention provides a one-component system thatwill be more quickly progressed through the regulatory approval process,allowing improved treatment methods to be translated to the clinic,where they are urgently needed.

[0230] E. Anti-Aminophospholipid Antibodies

[0231] E1. Polyclonal Anti-Aminophospholipid Antibodies

[0232] Means for preparing and characterizing antibodies are well knownin the art (see, e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; incorporated herein by reference). To preparepolyclonal antisera an animal is immunized with an immunogenicaminophospholipid composition, and antisera collected from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, mouse, rat, hamster, guinea pig or goat.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies.

[0233] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the present aminophospholipid immunogen; subcutaneous,intramuscular, intradermal, intravenous, intraperitoneal andintrasplenic. The production of polyclonal antibodies may be monitoredby sampling blood of the immunized animal at various points followingimmunization. A second, booster injection, may also be given. Theprocess of boosting and titering is repeated until a suitable titer isachieved. When a desired titer level is obtained, the immunized animalcan be bled and the serum isolated and stored. The animal can also beused to generate monoclonal antibodies.

[0234] As is well known in the art, the immolltogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Exemplary adjuvants includecomplete Freund's adjuvant, a non-specific stimulator of the immuneresponse containing killed Mycobacterilun tuberculosis; incompleteFreund's adjuvant; and aluminum hydroxide adjuvant.

[0235] It may also be desired to boost the host immune system, as may beachieved by associating aminophospholipids with, or couplingaminophospholipids to, a carrier. Exemplary carriers are keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers.

[0236] As is also known in the art, a given composition may vary in itsimmunogenicity. However, the generation of antibodies againstaminophospholipids is not particularly difficult. For example, highlyspecific anti-phosphatidylserine antibodies were raised in rabbitsimmunized by intramuscular injections of phosphatidylserine-containingpolyacrylamide gels and with phosphatidylserine-cytochrome c vesicles(Maneta-Peyret et al., 1988; 1989; each incorporated herein byreference). The use of acrylamide implants enhanced the production ofantibodies (Maneta-Peyret et al., 1988; 1989). Theanti-phosphatidylserine antibodies raised in this manner are able todetect phosphatidylserine in situ on human platelets (Maneta-Peyret etal., 1988). The groups of Inoue, Rote and Rauch have also developedanti-PS and anti-PE antibodies (see below).

[0237] E2. Monoclonal Anti-Aminophospholipid Antibodies

[0238] Various methods for generating monoclonal antibodies (MAbs) arealso now very well known in the art. The most standard monoclonalantibody generation techniques generally begin along the same lines asthose for preparing polyclonal antibodies (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory 1988; incorporated herein byreference). A polyclonal antibody response is initiated by immunizing ananimal with an immunogenic aminophospholipid composition and, when adesired titer level is obtained the immunized animal can be used togenerate MAbs.

[0239] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with the selected aminophospholipidimmunogen composition. The immunizing composition is administered in amanner effective to stimulate antibody producing cells. Rodents such asmice and rats are preferred animals, however, the use of rabbit, sheepand frog cells is also possible. The use of rats may provide certainadvantages (Goding, 1986, pp. 60-61; incorporated herein by reference),but mice are preferred, with the BALB/c mouse being most preferred asthis is most routinely used and generally gives a higher percentage ofstable fus′ions.

[0240] Following immunization, somatic cells with the potential forproducing aminophospholipid antibodies, specifically B lymphocytes (Bcells), are selected for use in the MAb generating protocol. These cellsmay be obtained from biopsied spleens, tonsils or lymph nodes, or from aperipheral blood sample. Spleen cells and peripheral blood cells arepreferred, the former because they are a rich source ofantibody-producing cells that are in the dividing plasmablast stage, andthe latter because peripheral blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

[0241] The anti-aminophospholipid antibody-producing B lymphocytes fromthe immunized animal are then fused with cells of an immortal myelomacell, generally one of the same species as the animal that wasimmunized. Myeloma cell lines suited for use in hybridoma-producingfusion procedures preferably are non-antibody-producing, have highfusion efficiency, and enzyme deficiencies that render then incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas).

[0242] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984; each incorporated herein by reference). For example, where theimmunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX1 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F,4B210 or one of the above listed mouse cell lines; and U-266,GMI500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful in connectionwith human cell fusions.

[0243] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 4:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976; each incorporated herein by reference),and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, byGefter et al. (1977; incorporated herein by reference). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986; incorporated herein by reference).

[0244] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10^(−8.) However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0245] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanithine phosphoribosyl transferase (IIPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0246] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired anti-aminophospholipidreactivity. The assay should be sensitive, simple and rapid, such asradioimmunoassays, enzyme inrmunoassays, cytotoxicity assays, plaqueassays, dot immunobinding assays, and the like.

[0247] The selected hybridomas would then be serially diluted and clonedinto individual anti-aminophospholipid antibody-producing cell lines,which clones can then be propagated indefinitely to provide MAbs. Thecell lines may be exploited for MAb production in two basic ways. Asample of the hybridoma can be injected (often into the peritonealcavity) into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. The individual cell lines could also becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

[0248] MAbs produced by either means will generally be further purified,e.g., using filtration, centrifugation and various chromatographicmethods, such as HPLC or affinity chromatography, all of whichpurification techniques are well known to those of skill in the art.These purification techniques each involve fractionation to separate thedesired antibody from other components of a mixture. Analytical methodsparticularly suited to the preparation of antibodies include, forexample, protein A-Sepharose and/or protein G-Sepharosc chromatography.

[0249] Umeda el al. (1989; incorporated herein by reference) reportedthe effective production of monoclonal antibodies recognizingstereo-specific epitopes of phosphatidylserine. The Umeda system isbased on the direct immunization of phosphatidylserine into mouse spleenusing a Salmonella-coated aminophospholipid sample (Umeda et al, 1989;incorporated herein by reference). The Umeda protocol gives a highfrequency of anti-PS MAbs, which exhibit three distinct reactivityprofiles ranging from highly specific to broadly cross-reactive. Umedais therefore also incorporated herein by reference for purposes offurther describing screening assays to identify MAbs that bindspecifically to PS, e.g., and do not bind to phosphatidylcholine.

[0250] Any of the 61 hybridomas generated by Umeda could potentially beemployed in the present invention. Examples are PSC8, PSF11, PSG3,PSD11, PSF10, PS1B, PS3D12, PS2C11; PS3A, PSF6, PSF7, PSB4, PS3H1; PS4A7and PS1G3. More preferred are PS3A, PSF6, PSF7, PSB4 and PS3H1 as theybind only to phosphatidylserine and phosphatidylethanolamine. Preferredanti-PS antibodies are PS4A7 (IgM) and PS1G3 (IgG₃), as they are highlyspecific for PS and exhibit no cross-reaction with other phospholipids.PS4A7 recognizes the stereo-specific configuration of the serine residuein PS (FIG. 1 Umeda et al., 1989; incorporated herein by reference).

[0251] Igarashi et al. (1991; incorporated herein by reference) alsoreported the effective induction of anti-PS antibodies of the IgGisotype by intrasplenic immunization. Only a slight increase of thetiter was observed when the antigen was again injected intravenously. Ahigh frequency of anti-PS MAbs of the IgG isotype was also observed evenwhen MAbs were produced 10 days after the intrasplenic injection of theantigen. These antibodies were also employed by Schuurmans Stekhoven etal. (1994).

[0252] The other significant anti-PS antibody production has been byRote and colleagues. Rote et al. (1993; incorporated herein byreference) particularly employed PS micelles in combination withFreund's complete adjuvant to generate specific anti-PS antibodies. Roteel al. (1993) also generated monoclonal antibodies that differentiatebetween cardiolipin (CL) and PS. Rote et al. (1993) is therefore alsoincorporated herein by reference for purposes of further describingscreening assays to identify MAbs that bind specifically to PS bytesting against resting and thrombin-activated platelets using flowcytometry.

[0253] The 3SB9b antibody produced by Rote et al. (1993) reacted withonly with PS, and is a preferred antibody for use in the presentinvention. BA3B5C4 may also be used as it reacts with both PS and CL.These antibodies are also described in Lin et al. (1995), Obringer etal. (1995) and Katsuragawa et al. (1997).

[0254] E3. Anti-Aminophospholipid Antibodies from Phagemid Libraries

[0255] Recombinant technology now allows the preparation of antibodieshaving the desired specificity from recombinant genes encoding a rangeof antibodies (Van Dijk et al., 1989; incorporated herein by reference).Certain recombinant techniques involve the isolation of the antibodygenes by immunological screening of combinatorial immunoglobulin phageexpression libraries prepared from RNA isolated from the spleen of animmunized animal (Morrison et al., 1986; Winter and Milstein, 1991; eachincorporated herein by reference).

[0256] For such methods, combinatorial immunoglobulin phagemid librariesare prepared from RNA isolated from the spleen of the immunized animal,and phagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 1IO times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination, which further increases the percentage of appropriateantibodies generated.

[0257] One method for the generation of a large repertoire of diverseantibody molecules in bacteria utilizes the bacteriophage lambda as thevector (lHuse et al., 1989; incorporated herein by reference).Production of antibodies using the lambda vector involves the cloning ofheavy and light chain populations of DNA sequences into separatestarting vectors. The vectors are subsequently combined randomly to forma single vector that directs the co-expression of heavy and light chainsto form antibody fragments. The heavy and light chain DNA sequences areobtained by amplification, preferably by PCR™ or a related amplificationtechnique, of mRNA isolated from spleen cells (or hybridomas thereof)from an animal that has been immunized with a selected antigen. Theheavy and light chain sequences are typically amplified using primersthat incorporate restriction sites into the ends of the amplified DNAsegment to facilitate cloning of the heavy and light chain segments intothe starting vectors.

[0258] Another method for the generation and screening of largelibraries of wholly or partially synthetic antibody combining sites, orparatopes, utilizes display vectors derived from filamentous phage suchas M13, fl or fd. These filamentous phage display vectors, referred toas “phagemids”, yield large libraries of monoclonal antibodies havingdiverse and novel immunospecificities. The technology uses a filamentousphage coat protein membrane anchor domain as a means for linkinggene-product and gene during the assembly stage of filamentous phagereplication, and has been used for the cloning and expression ofantibodies from combinatorial libraries (Kang et al., 1991; Barbas etal., 1991; each incorporated herein by reference).

[0259] This general technique for filamentous phage display is describedin U.S. Pat. No. 5,658,727, incorporated herein by reference. In a mostgeneral sense, the method provides a system for the simultaneous cloningand screening of pre-selected ligand-binding specificities from antibodygene repertoires using a single vector system. Screening of isolatedmembers of the library for a pre-selected ligand-binding capacity allowsthe correlation of the binding capacity of an expressed antibodymolecule with a convenient means to isolate the gene that encodes themember from the library.

[0260] Linkage of expression and screening is accomplished by thecombination of targeting of a fusion polypeptide into the periplasm of abacterial cell to allow assembly of a functional antibody, and thetargeting of a fusion polypeptide onto the coat of a filamentous phageparticle during phage assembly to allow for convenient screening of thelibrary member of interest. Periplasmic targeting is provided by thepresence of a secretion signal domain in a fusion polypeptide. Targetingto a phage particle is provided by the presence of a filamentous phagecoat protein membrane anchor domain (i.e., a cpIII- or cpVIII-derivedmembrane anchor domain) in a fusion polypeptide.

[0261] The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chaingenes, by altering one or more of the complementarity determiningregions of the cloned heavy chain genes of the library, or byintroducing random mutations into the library by error-prone polymerasechain reactions. Additional methods for screening phagemid libraries aredescribed in U.S. Pat. Nos. 5,580,717; 5,427,908; 5,403,484; and5,223,409, each incorporated herein by reference.

[0262] Another method for the screening of large combinatorial antibodylibraries has been developed, utilizing expression of populations ofdiverse heavy and light chain sequences on the surface of a filamentousbacteriophage, such as M13, fl or fd (U.S. Pat. No. 5,698,426;incorporated herein by reference). Two populations of diverse heavy (Hc)and light (Lc) chain sequences are synthesized by polymerase chainreaction (PCR™). These populations are cloned into separate M13-basedvector containing elements necessary for expression. The heavy chainvector contains a gene VIII (gVIII) coat protein sequence so thattranslation of the heavy chain sequences produces gVIII-Hc fusionproteins. The populations of two vectors are randomly combined such thatonly the vector portions containing the Hc and Lc sequences are joinedinto a single circular vector.

[0263] The combined vector directs the co-expression of both Hc and Lcsequences for assembly of the two polypeptides and surface expression onM13 (U.S. Pat. No. 5,698,426; incorporated herein by reference). Thecombining step randomly brings together different lic and Lc encodingsequences within two diverse populations into a single vector. Thevector sequences donated from each independent vector are necessary forproduction of viable phage. Also, since the pseudo gVIII sequences arecontained in only one of the two starting, vectors, co-expression offunctional antibody fragments as Lc associated gVIII-Hc fusion proteinscannot be accomplished on the phage surface until the vector sequencesare linked in the single vector.

[0264] Surface expression of the antibody library is performed in anamber suppressor strain. An amber stop codon between the He sequence andthe gVIII sequence unlinks the two components in a non-suppressorstrain. Isolating the phage produced from the non-suppressor strain andinfecting a suppressor strain will link the He sequences to the gVIIIsequence during expression. Culturing the suppressor strain afterinfection allows the coexpression on the surface of M13 of all antibodyspecies within the library as gVIII fusion proteins (gVIII-Fab fusionproteins). Alternatively, the DNA can be isolated from thenon-suppressor strain and then introduced into a suppressor strain toaccomplish the same effect.

[0265] The surface expression library is screened for specific Fabfragments that bind preselected molecules by standard affinity isolationprocedures. Such methods include, for example, panning (Parmley andSmith, 1988; incorporated herein by reference), affinity chromatographyand solid phase blotting procedures. Panning is preferred, because hightiters of phage can be screened easily, quickly and in small volumes.Furthermore, this procedure can select minor Fab fragments specieswithin the population, which otherwise would have been undetectable, andamplified to substantially homogenous populations. The selected Fabfragments can be characterized by sequencing the nucleic acids encodingthe polypeptides after amplification of the phage population.

[0266] Another method for producing diverse libraries of antibodies andscreening for desirable binding specificities is described in U.S. Pat.No. 5,667,988 and U.S. Pat. No. 5,759,817, each incorporated herein byreference. The method involves the preparation of libraries ofheterodimeric immunoglobulin molecules in the form of phagemid librariesusing degenerate oligonucleotides and primer extension reactions toincorporate the degeneracies into the CDR regions of the immunoglobulinvariable heavy and light chain variable domains, and display of themutagelizcd polypeptides on the surface of the phagemid. Thereafter, thedisplay protein is screened for the ability to bind to a preselectedantigen.

[0267] The method for producing a heterodimeric immunoglobulin moleculegenerally involves (1) introducing a heavy or light chain Vregion-coding gene of interest into the phagemid display vector; (2)introducing a randomized binding site into the phagemid display proteinvector by primer extension with an oligonucleotide containing regions ofhomology to a CDR of the antibody V region gene and containing regionsof degeneracy for producing randomized coding sequences to form a largepopulation of display vectors each capable of expressing differentputative binding sites displayed on a phagemid surface display protein;(3) expressing the display protein and binding site on the surface of afilamentous phage particle; and (4) isolating (screening) thesurface-expressed phage particle using affinity techniques such aspanning of phage particles against a preselected antigen, therebyisolating one or more species of phagemid containing a display proteincontaining a binding site that binds a preselected antigen.

[0268] A further variation of this method for producing diverselibraries of antibodies and screening for desirable bindingspecificities is described in U.S. Pat. No. 5,702,892, incorporatedherein by reference. In this method, only heavy chain sequences areemployed, the heavy chain sequences are randomized at all nucleotidepositions which encode either the CDRJ or CDRIII hypervariable region,and the genetic variability in the CDRs is generated independent of anybiological process.

[0269] In the method, two libraries are engineered to geneticallyshuffle oligonucleotide motifs within the framework of the heavy chaingene structure. Through random mutation of either CDRI or CDRIII, thehypervariable regions of the heavy chain gene were reconstructed toresult in a collection of highly diverse sequences. The heavy chainproteins encoded by the collection of mutated gene sequences possessedthe potential to have all of the binding characteristics of animmunoglobulin while requiring only one of the two immunoglobulinchains.

[0270] Specifically, the method is practiced in the absence of theimmuLnoglobLlin light chain protein. A library of phage displayingmodified heavy chain proteins is incubated with an immobilized ligaindto select clones encoding recombinant proteins that specifically bindthe immobilized ligand. The bound phage are then dissociated from theimmobilized ligand and amplified by growth in bacterial host cells.Individual viral plaques, each expressing a different recombinantprotein, are expanded, and individual clones can then be assayed forbinding activity.

[0271] E4. Anti-Aminophospholipid Antibodies from Human Patients

[0272] Antibodies against aminophospholipids, particularlyphosphatidylserine and phosphatidylethanolamine, occur in the humanpopulation, where they are correlated with certain disease states.Anti-aminophospholipid antibodies are part of the heterogeneousanti-phospholipid antibodies (aPL), observed to have families ofdifferent specificities and classes. Primary anti-phospholipid syndrome(APS) has even been separated from other forms of autoimmune diseaseassociated with anti-phospholipid antibody production.

[0273] Anti-PS antibodies are particularly associated with recurrentpregnancy loss (Rote et al., 1995; Rote, 1996; Vogt et al., 1996; Vogtet al., 1997; incorporated herein by reference) and with the autoimmunedisease, systemic lupus erythematosus (SLE or “lupus”) (Branch et al.,1987; incorporated herein by reference). Anti-PE antibodies have alsobeen reported in human patients, particularly those with autoimmunediseases (Staub et al., 1989). Branch et al. (1987) reported that 80% ofpatients with lupus anticoagulant (LA or LAC) had autoantibodies thatrecognized PE; with Drouvalakis and Buchanan (1998) increasing thisnumber to 95% PE-positives from autoimmune LAC sera.

[0274] Anti-phospholipid antibodies are not to be confused withanti-endothelial cell antibodies (AECA), although they can be found inthe same patient. The existence of AECA has been documented in a varietyof clinical settings associated with vasculitis, such as systemicsclerosis (SS). To study AECA, antibodies are obtained from patientsthat do not have anti-phospholipid antibodies (aPL-negative sera).

[0275] The pathogenic role of AECA remains unclear, although Bordron etal. (1998) very recently suggested that AECA may initiate apoptosis inendothelial cells, which would be followed by PS transfer to the outerface of the membrane. They proposed that this would account for thesubsequent generation of the anti-phospholipid antibodies that aresometimes seen in conjunction with AECA in patients with skin lesions orconnective tissue disease (Bordron et al., 1998). However, although AECAbinding to an apoptosis-inducing antigen was postulated, these studiesdid not lead to the further characterization of AECA, still said torepresent an extremely heterogeneous family of antibodies reacting withdifferent (non-lipid) structures on endothelial cells (Bordron et al.,1998).

[0276] Anti-phosphatidylserine antibodies are closely associated withpregnancy loss, pregnancy-induced hypertension and intrauterine growthretardation. A phosphatidylserine-dependent antigen has been shown to beexpressed on the surface of a choriocarcinoma model .2.: (BeWo) ofdifferentiating cytotrophoblastic cells, indicating that it should beaccessible in vivo to circulating anti-phosphatidylserine antibodies(Rote et al., 1995). Indeed, Vogt et al. (1996) showed that themonoclonal antibody 3SB9b, which reacts with phosphatidylserine but notcardiolipin, induced a significant reduction in both fet al andplacental weights in a mouse model for the anti-phospholipid antibodysyndrome

[0277] These authors developed a model for explaining miscarriagesassociated with anti-phospholipid antibodies: anti-phosphatidylserineantibody reveals sites for prothrombin binding on the surface of thetrophoblast, most likely by removing Annexin V (Vogt et al., 1997).Trophoblast differentiation is associated with externalization ofphosphatidylserine from the inner to the outer surface of the plasmamembrane. Normally, externalization of phosphatidylserine is concurrentwith binding of Anmexin V, which prevents the phosphatidylserine-richsurface from acting as a site for activation of coagulation. Thus, whenanti-phospholipid antibodies are present. they prevent Annexin V bindingand lead to a procoagulant state (Vogt el al., 1997).

[0278] Anti-PE antibodies are frequently associated with Ilipusanticoagulants (LAC sera). The role of PE and anti-PE in LAC isextremely complex, see, e.g., Smirnov el al. (1995; incorporated hereinby reference), where various hypotheses are set forth. Smirnov et al(1995) report that, in the presence of activated protein C and PE, LACplasma clots faster than normal plasma. Rauch et al. (1986) characterizeLAC anti-phospholipid antibodies as prolonging the clotting time in invitro coagulation assays.

[0279] Vlachoyiannopoulos et al. (1993; incorporated herein byreference) tested SLE and APS sera by ELISA for antibodies tophosphatidylethanolamine and cardiolipin, as compared to healthy blooddonors. Both SLE and APS patients were reported to present a highertiter of IgM anti-PE antibodies than normal subjects, while the IgG andIgA anti-PE reactivity reportedly did not differ. It was suggested thatIgA and IgG anti-PE antibodies may occur in low titers as naturalautoantibodies in normal subjects (Vlachoyiannopoulos et al., 1993;incorporated herein by reference).

[0280] Rauch et al. (1986; incorporated herein by reference) producedhybridomas by fusing lymphocytes from 13 systemic lupus erythematosuspatients with a lymphoblastoid line. They demonstrated that theautoantibodies that prolonged clotting time bound to hexagonal phasephospholipids, including natural and synthetic forms ofphosphatidylethanolamine (Rauch et al., 1986; incorporated herein byreference). In contrast, lamellar phospholipids, such asphosphatidylcholine and synthetic lamellar forms ofphosphatidylethanolamine, had no effect on the anticoagulant activity(Rauch et al., 1986).

[0281] Rauch and Janoff (1990; incorporated herein by reference) went onto show that immunization of mice with phosphatidylethanolamine in thehexagonal II phase, but not in the bilayer phase, resulted in theinduction of anti-phospholipid antibodies. These antibodies werestrongly reactive with phosphatidylethanolamine and had functional lupusanticoagulant activity characteristic of autoantibodies from patientswith autoimmiiiiune disease (Ranch and Janoff, 1990).

[0282] The hexagonal II phase form of aminophospholipids should thus beadvantageously used to generate antibodies for use in the presentinvention. Indeed. Trudell reported that antibodies raised against TFA-(trifluoroacetyl-) protein adducts bind to TFA-phosphatidylethanolaminein hexagonal phase phospholipid micelles, but not in lamellar liposomes(Trudell et al., 1991a; incorporated herein by reference). The authorssuggested that TFA-phosphatidylethanolamine adducts that reside innon-lamellar domains on the hepatocyte surface could be recognitionsites for anti-TFA-adduct antibodies and potentially participate inimmune-mediated halothane hepatotoxicity (Trudell et al., 1991a). It waslater shown that these same antibodies cross-react withTFA-dioleoylphosphatidylethanolamine when this adduct is incorporatedinto the surface of hepatocytes (Trudell et al., 1991b; incorporatedherein by reference), thus supporting this hypothesis.

[0283] Berard further explained the hexagonal II phase form ofaminophospholipids, such as PE (Berard et al., 1993; incorporated hereinby reference). In bilayers, phospholipids generally adopt a gelstructure, crystalline lattice or lamellar phase (Berard et al., 1993).However, depending on the cholesterol content, protein and ionicenvironments, phospholipids can easily change phases, adopting ahexagonal II phase (Berard et al., 1993; incorporated herein byreference). It is this hexagonal II phase of aminophospholipids that isbelieved to be immunogenic, as initial proposed for autoantibodygeneration in disease situations (Berard et al., 1993; incorporatedherein by reference).

[0284] Qamar et al. (1990; incorporated herein by reference) havedeveloped a variation on the hexagonal aminophospholipid recognitiontheme. Using phosphatidylethanolamine as a model, these authors reportedthat anti-PE antibodies from aPL-positive SLE sera do not bind to PE,but in fact are directed to lysophosphatidylethanolamine (1 PE), anatural PE degradation product and a likely contaminant of most PEpreparations (Qamar et al., 1990; incorporated herein by reference).

[0285] Other recent data indicate that most anti-phospholipid antibodiesrecognize phosplholipid in the context of nearby proteins (Rote, 1996;Chamley et al, 1991). In plasma membranes, the majority of thephospholipid appears to be naturally in non-antigenic bilaminar form(Rote, 1996). Accessory molecules may help facilitate the transition tohexagonal antigenic forms and stabilize their expression (Galli el al.,1993). For example, naturally occurring anti-phospholipid antibodieswere first reported to recognize complexes of cardiolipin orphosphatidylserine with β₂-glycoprotein I (β₂-GPI or apolipoprotein H,apoH) (Galli et al., 1990; 1993). β₂-GPI is believed to stabilizephospholipids in antigenic conformations that do not exist in purephospholipids (McNeil et al., 1990; U.S. Pat. No. 5,344,758; Chamley etal., 1991; Matsuura et al., 1994). Prothrombin has also been implicatedin the phospholipid stabilization process (Bevers et al., 1991).

[0286] Phospholipid-binding plasma proteins are also generally necessaryfor antibody recognition of the electrically neutral or zwitterionicphospholipid, phosphatidylethanolamine. Sugi and McIntyre (1995;incorporated herein by reference) identified two prominent PE-bindingplasma proteins as high molecular weight kininogen (HMWK or HK) and lowmolecular weight kininogen (LMWK or LK). Anti-PE antibodies frompatients with SLE and/or recurrent spontaneous abortions were shown notto recognize PE, HMWK or LMWK when they were presented independently assole antigens on ELISA plates (Sugi and McIntyre, 1995). Otheranti-PE-positive sera that did not react with PE-HMWK or PE-LMWK weresuggested to recognize factor XI or prekallikrein, which normally bindto HMWK (Sugi and McIntyre, 1995; incorporated herein by reference).

[0287] The validity of these results was confirmed by showing thatintact HMWK binds to various phospholipids, such as cardiolipin,phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine;but that anti-PE antibodies recognize only a kininogen-PE complex, anddo not recognize kininogens presented with other phospholipid substrates(Sugi and McIntyre, 1996a; incorporated herein by reference). Thisindicates that PE induces unique antigenic conformational changes in thekininogens that are not induced when the kininogens bind to otherphospholipids (SuLgi and McIntyre, 1996a).

[0288] It has further been suggested that kininogens can bind toplatelets by virtue of- exposed PE in the platelet membrane (Sugi andMcIntyre, 1996b; incorporated herein by reference). Exogenously addedkininogen-dependent anti-PE was shown to increase thrombin-inducedplatelet aggregation in vitro, but not to alter ADP-induced aggregation(Sugi and McIntyre, 1996b; incorporated herein by reference). Incontrast, kininogen independent anti-PE, which recognized PE per se, wasreported not augment thrombin-induced platelet aggregation. It was thusproposed that kininogen dependent anti-PE may disrupt the normalanti-thrombotic effects of kininogen (Sugi and McIntyre, 1996b;incorporated herein by reference).

[0289] Anti-aminophospholipid antibodies from human patients aretherefore a mixture of antibodies that generally recognizeaminophospholipids stabilized by protein interactions (Rote, 1996). Theantibodies may bind to stabilized phospholipid epitopes, or may bind toan epitope formed from the interaction of the phospholipid and aminoacids on the stabilizing protein (Rote, 1996). Either way, suchantibodies clearly recognize aminophospholipids in natural membranes inthe human body, probably associated with plasma proteins (McNeil et al.,1990; Bevers et al., 1991). These antibodies would thus be appropriateas starting materials for generating an antibody for use in the presentinvention.

[0290] To prepare an anti-aminophospholipid antibody from a humanpatient, one would simply obtain human lymphocytes from an individualhaving anti-aminophospholipid antibodies, for example from humanperipheral blood, spleen, lymph nodes, tonsils or the like, utilizingtechniques that are well known to those of skill in the art. The use ofperipheral blood lymphocytes will often be preferred.

[0291] Human monoclonal antibodies may be obtained from the humanlymphocytes producing the desired anti-aminophospholipid antibodies byimmortalizing the human lymphocytes, generally in the same manner asdescribed above for generating any monoclonal antibody. The reactivitiesof the antibodies in the culture supernatants are generally firstchecked, employing one or more selected aminoplhospholipid antigen(s),and the lymphocytes that exhibit high reactivity are grown. Theresulting lymphocytes are then fused with a parent line of human ormouse origin, and further selection gives the optimal clones.

[0292] The recovery of monoclonal antibodies from the immortalized cellsmay be achieved by any method generally employed in the production ofmonoclonal antibodies. For instance, the desired monoclonal antibody maybe obtained by cloning the immortalized lymphocyte by the limitingdilution method or the like, selecting the cell producing the desiredantibody, growing the selected cells in a medium or the abdominal cavityof an animal, and recovering the desired monoclonal antibody from theculture supernatant or ascites.

[0293] Such techniques have been used, for example, to isolate humanmonoclonal antibodies to Pseudomonas aeruginosa epitopes (U.S. Pat. Nos.5,196,337 and 5,252,480, each incorporated herein by reference);polyribosylribitol phosphate capsular polysaccharides (U.S. Pat. No.4,954,449, incorporated herein by reference); the Rh(D) antigen (U.S.Pat. No. 5,665,356, incorporated herein by reference); and viruses, suchas human immunodeficiency virus, respiratory syncytial virus, herpessimplex virus, varicella zoster virus and cytomegalovirus (U.S. Pat.Nos. 5,652,138; 5,762,905; and 4,950,595, each incorporated herein byreference).

[0294] The applicability of the foregoing techniques to the generationof human anti-aminophospholipid antibodies is clear. Rauch et al. (1986;incorporated herein by reference) generally used such methods to producehybridomas by fusing lymphocytes from SLE patients with a lymphoblastoidline. This produced human antibodies that bound to hexagonal phasephospholipids, including natural and synthetic forms ofphosphatidylethanolamine (Rauch et al, 1986; incorporated herein byreference).

[0295] Additionally, the methods described in U.S. Pat. No. 5,648,077(incorporated herein by reference) can be used to form a trioma or aquadroma that produces a human antibody against a selectedaminophospholipid. In a general sense, a hybridoma cell line comprisinga parent rodent immortalizing cell, such as a murine myeloma cell, e.g.SP-2, is fused to a human partner cell, resulting in an immortalizingxenogeneic hybridoma cell. This xenogeneic hybridoma cell is fused to acell capable of producing an anti-aminophospholipid human antibody,resulting in a trioma cell line capable of generating human antibodyeffective against such antigen in a human. Alternately, when greaterstability is desired, a trioma cell line which preferably no longer hasthe capability of producing its own antibody is made, and this trioma isthen fused with a further cell capable of producing an antibody usefulagainst the aminophospholipid antigen to obtain a still more stablehybridoma (quadroma) that produces antibody against the antigen.

[0296] E5. Anti-Aminophospholipid Antibodies from Human Lymphocytes

[0297] In vitro immunization, or antigen stimulation, may also be usedto generate a human anti-aminophospholipid antibody. Such techniques canbe used to stimulate peripheral blood lymphocytes from bothanti-aminophospholipid antibody-producing human patients, and also fromnormal, healthy subjects. Indeed, Vlachoyiannopoulos et al. (1993;incorporated herein by reference) reported that low titeranti-aminophospholipid antibodies occur in normal subjects. Even if thiswere not the case, anti-aminophospholipid antibodies can be preparedfrom healthy human subjects, simply by stimulating antibody-producingcells with aminophospholipids in vitro.

[0298] Such “in vitro immunization” involves antigen-specific activationof non-immunized B lymphocytes, generally within a mixed population oflymphocytes (mixed lymphocyte cultures, MLC). In vitro immunizations mayalso be supported by B cell growth and differentiation factors andlymphokines. The antibodies produced by these methods are often IgMantibodies (Borrebaeck et al., 1986; incorporated herein by reference).

[0299] Another method has been described (U.S. Pat. No. 5,681,729,incorporated herein by reference) wherein human lymphocytes that mainlyproduce IgG (or IgA) antibodies can be obtained. The method involves, ina general sense, transplanting human lymphocytes to an immunodeficientanimal so that the human lymphocytes “take” in the animal body;immunizing the animal with a desired antigen, so as to generate humanlymphocytes producing an antibody specific to the antigen; andrecovering the human lymphocytes producing the antibody from the animal.The human lymphocytes thus produced can be used to produce ai monoclonalantibody by immortalizing the human lymphocytes producing the antibody.cloning the obtained immortalized human-originated lymphocytes producingthe antibody, and recovering a monoclonal antibody specific to thedesired antigen from the cloned immortalized human-originatedlymphocytes.

[0300] The immunodeficient animals that may be employed in thistechnique are those that do not exhibit rejection when human lymphocytesare transplanted to the animals. Such animals may be artificiallyprepared by physical, chemical or biological treatments. Anyimmunodeficient animal may be employed. The human lymphocytes may beobtained from human peripheral blood, spleen, lymph nodes, tonsils orthe like.

[0301] The “taking” of the transplanted human lymphocytes in the animalscan be attained by merely administering the human lymphocytes to theanimals. The administration route is not restricted and may be, forexample, subcutaneous, intravenous or intraperitoneal. The dose of thehuman lymphocytes is not restricted, and can usually be 10⁶ to 10⁸lymphocytes per animal. The immunodeficient animal is then immunizedwith the desired aminophospholipid antigen.

[0302] After the immunization, human lymphocytes are recovered from theblood, spleen, lymph nodes or other lymphatic tissues by anyconventional method. For example, mononuclear cells can be separated bythe Ficoll-Hypaque (specific gravity: 1.077) centrifugation method, andthe monocytes removed by the plastic dish adsorption method. Thecontaminating cells originating from the inimunodeficient animal may beremoved by using an antiserum specific to the animal cells. Theantiserum may be obtained by, for example, inimunizing a second,distinct animal with the spleen cells of the immunodeficient animal, andrecovering serum from the distinct immunized animal. The treatment withthe antiserum may be carried out at any stage. The human lymphocytes mayalso be recovered by an immunological method employing a humanimmunoglobulin expressed on the cell surface as a marker.

[0303] By these methods, human lymphocytes mainly producing IgG and IgAantibodies specific to one or more selected aminophospholipid(s) can beobtained. Monoclonal antibodies are then obtained from the humanlymphocytes by immortalization, selection, cell growth and antibodyproduction.

[0304] E6. Transgenic Mice Containing Human Antibody Libraries

[0305] Recombinant technology is now available for the preparation ofantibodies. In addition to the combinatorial immunoglobulin phageexpression libraries disclosed above, another molecular cloning approachis to prepare antibodies from transgenic mice containing human antibodylibraries. Such techniques are described in U.S. Pat. No. 5,545,807,incorporated herein by reference.

[0306] In a most general sense, these methods involve the production ofa transgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material may be produced from a human source, or may beproduced synthetically. The material may code for at least part of aknown immunoglobulin or may be modified to code for at least part of analtered immunoglobulin.

[0307] The inserted genetic material is expressed in the transgenicanimal, resulting in production of an immunoglobulin derived at least inpart from the inserted human immunoglobulin genetic material. It isfound the genetic material is rearranged in the transgenic animal, sothat a repertoire of immunoglobulins with part or parts derived frominserted genetic material may be produced, even if the inserted geneticmaterial is incorporated in the geriline in the wrong position or withthe wrong geometry.

[0308] The inserted genetic material may be in the form of DNA clonedinto prokaryotic vectors such as plasmids and/or cosmids. Larger DNAfragments are inserted using yeast artificial chromosome vectors (Burkeet al., 1987; incorporated herein by reference), or by introduction ofchromosome fragments (Richer and Lo, 1989; incorporated herein byreference). The inserted genetic material may be introduced to the hostin conventional manner, for example by injection or other proceduresinto fertilized cgg,s or embryonic stem cells.

[0309] In preferred aspects, a host animal that initially does not carrygenetic material encoding immunoglobulini constant regions is utilized,so that the resulting transgenic animal will use only the inserted humangenetic material when producing immunoglobulins. This can be achievedeither by using a naturally occurring mutant host lacking the relevantgenetic material, or by artificially making mutants e g., in cell linesultimately to create a host from which the relevant genetic material hasbeen removed.

[0310] Where the host animal carries genetic material encodingimmunoglobulin constant regions, the transgenic animal will carry thenaturally occurring genetic material and the inserted genetic materialand will produce immunoglobulins derived from the naturally occurringgenetic material, the inserted genetic material, and mixtures of bothtypes of genetic material. In this case the desired immunoglobulin canbe obtained by screening hybridomas derived from the transgenic animal,e.g., by exploiting the phenomenon of allelic exclusion of antibody geneexpression or differential chromosome loss.

[0311] Once a suitable transgenic animal has been prepared, the animalis simply immunized with the desired immunogen. Depending on the natureof the inserted material, the animal may produce a chimericimmunoglobulin, e.g. of mixed mouse/human origin, where the geneticmaterial of foreign origin encodes only part of the immunoglobulin; orthe animal may produce an entirely foreign immunoglobulin, e.g. ofwholly human origin, where the genetic material of foreign originencodes an entire immunoglobulin.

[0312] Polyclonal antisera may be produced from the transgenic animalfollowing immunization. Immunoglobulin-producing cells may be removedfrom the animal to produce the immunoglobulin of interest. Preferably,monoclonal antibodies are produced from the transgenic animal, e.g., byfusing spleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

[0313] In an alternative approach, the genetic material may beincorporated in the animal in such a way that the desired antibody isproduced in body fluids such as serum or external secretions of theanimal, such as milk, colostrum or saliva. For example, by inserting invitro genetic material encoding for at least part of a humanimmunoglobulin into a gene of a mammal coding for a milk protein andthen introducing the gene to a fertilized egg of the mammal, e.g., byinjection, the egg may develop into an adult female mammal producingmilk containing immunoglobulin derived at least in part from theinserted human immunoglobulin genetic material. The desired antibody canthen be harvested from the milk. Suitable techniques for carrying outsuch processes are known to those skilled in the art.

[0314] The foregoing transgenic animals are usually employed to producehuman antibodies of a single isotype, more specifically an isotype thatis essential for B cell maturation, such as IgM and possibly IgD.Another preferred method for producing human anti-aminophospholipidantibodies is described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429; each incorporated byreference, wherein transgenic animals are described that are capable ofswitching from an isotype needed for B cell development to otherisotypes.

[0315] In the development of a B lymphocyte, the cell initially producesIgM with a binding specificity determined by the productively rearrangedVH and VL regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain. The use of mu or delta constantregions is largely determined by alternate splicing, permitting IgM andIgD to be coexpressed in a single cell. The other heavy chain isotypes(gamma, alpha, and epsilon) are only expressed natively after a generearrangement event deletes the C mu and C delta exons. This generearrangement process, tenrned isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except delta). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences.

[0316] For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1-2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5′ flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. Although a 5′ flanking sequencefrom one switch region can be operably linked to a different switchregion for transgene construction, in some embodiments it is preferredthat each switch region incorporated in the transgene construct have the5′ flanking region that occurs immediately upstream in the naturallyoccurring gerniline configuration. Sequence information relating toimmunoglobulin switch region sequences is known (Mills et al., 1990;Sideras et al., 1989; each incorporated herein by reference).

[0317] In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, the human immunoglobulintransgenes contained within the transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

[0318] An important requirement for transgene function is the generationof a primary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmernbrane and secreted immunioglobullis.

[0319] Fhe human heavy chain locus consists of approximately 200 V genesegments spanning 2 Mb, approximately 30)D gene segments spanning about40 kb, six J segments clustered within a 3 kb span, and nine constantregion gene segments spread out over approximately 300 kb. The entirelocus spans approximately 2.5 Mb of the distal portion of the long armof chromosome 14. Heavy chain transgene fragments containing members ofall six of the known V_(H) families, the D and J gene segments, as wellas the mu, delta, gamma 3, gamma 1 and alpha 1 constant regions areknown (Berman et al., 1988; incorporated herein by reference). Genomicfragments containing all of the necessary gene segments and regulatorysequences from a human light chain locus is similarly constructed.

[0320] The expression of successfully rearranged immunoglobulin heavyand light transgenes usually has a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, in certain embodiments, it is desirable toeffect complete inactivation of the endogenous Ig loci so that hybridimmunoglobulin chains comprising a human variable region and a non-human(e.g., murine) constant region cannot be formed, for example bytrans-switching between the transgene and endogenous Ig sequences. Usingembryonic stem cell technology and homologous recombination, theendogenous immunoglobulin repertoire can be readily eliminated. Inaddition, suppression of endogenous Ig genes may be accomplished using avariety of techniques, such as antisense technology.

[0321] In other aspects of the invention, it may be desirable to producea trans-switched immunoglobulin. Antibodies comprising such chimerictrans-switched immunoglobulins can be used for a variety of applicationswhere it is desirable to have a non-human (e.g., murine) constantregion, e.g., for retention of effector functions in the host. Thepresence of a murine constant region can afford advantages over a humanconstant region, for example, to provide murine effector functions (eg., ADCC, murine complement fixation) so that such a chimeric antibodymay be tested in a mouse disease model. Subsequent to the animaltesting, the human variable region encoding sequence may be isolated,e.g., by PCR amplification or cDNA cloning from the source (hybridomaclone), and spliced to a sequence encoding a desired human constantregion to encode a human sequence antibody more suitable for humantherapeutic use.

[0322] E7. Humanized Anti-Aminophospholipid Antibodies

[0323] Human antibodies generally have at least three potentialadvantages for use in human therapy. First, because the effector portionis human, it may interact better with the other parts of the humanimmune system, e.g., to destroy target cells more efficiently bycomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC). Second, the human immune system should notrecognize the antibody as foreign. Third, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given.

[0324] Various methods for preparing human anti-aminophospholipids areprovided herein. In addition to human antibodies, “humanized” antibodieshave many advantages. “Humanized” antibodies are generally chimeric ormutant monoclonal antibodies from mouse, rat, hamster, rabbit or otherspecies, bearing human constant and/or variable region domains orspecific changes. Techniques for generating a so-called “humanized”anti-aminophospholipid antibody are well known to those of skill in theart.

[0325] Humanized antibodies also share the foregoing advantages. First,the effector portion is still human. Second, the human immune systemshould not recognize the framework or constant region as foreign, andtherefore the antibody response against such an injected antibody shouldbe less than against a totally foreign mouse antibody. Third, injectedhumanized antibodies, as opposed to injected mouse antibodies, willpresumably have a half-life more similar to naturally occurring humanantibodies, also allowing smaller and less frequent doses.

[0326] A number of methods have been described to produce humanizedantibodies. Controlled rearrangement of antibody domains joined throughprotein diSulfide bonds to form new, artificial protein molecules or“chimeric” antibodies can be utilized (Konieczny et al., 1981;incorporated herein by reference). Recombinant DNA technology can alsobe used to construct gene fusions between DNA sequences encoding mouseantibody variable light and heavy chain domains and human antibody lightand heavy chain constant domains (Morrison et al., 1984; incorporatedherein by reference).

[0327] DNA sequences encoding the antigen binding portions orcomplementarity determining regions (CDR's) of murine monoclonalantibodies can be grafted by molecular means into the DNA sequencesencoding the frameworks of human antibody heavy and light chains (Joneset al., 1986; Riechimann et al., 1988; each incorporated herein byreference). The expressed recombinant products are called “reshaped” orhumanized antibodies, and comprise the framework of a human antibodylight or heavy chain and the antigen recognition portions, CDR's, of amurine monoclonal antibody.

[0328] Another method for producing humanized antibodies is described inU.S. Pat. No. 5,639,641, incorporated herein by reference. The methodprovides, via resurfacing, humanized rodent antibodies that haveimproved therapeutic efficacy due to the presentation of a human surfacein the variable region. In the method: (1) position alignments of a poolof antibody heavy and light chain variable regions is generated to givea set of heavy and light chain variable region framework surface exposedpositions, wherein the alignment positions for all variable regions areat least about 98% identical; (2) a set of heavy and light chainvariable region framework surface exposed amino acid residues is definedfor a rodent antibody (or fragment thereof); (3) a set of heavy andlight chain variable region framework surface exposed amino acidresidues that is most closely identical to the set of rodent surfaceexposed amino acid residues is identified; (4) the set of heavy andlight chain variable region framework surface exposed amino acidresidues defined in step (2) is substituted with the set of heavy andlight chain variable region framework surface exposed amino acidresidues identified in step (3), except for those amino acid residuesthat are within 5A of any atom of any residue of the complemiienitaritydetermining regions of the rodent antibody; and (5) the humanized rodentantibody having binding specificity is produced.

[0329] A similar method for the production of humanized antibodies isdescribed in U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and5,530,101, each incorporated herein by reference. These methods involveproducing humanized immunoglobulins having one or more complementaritydetermining regions (CDR's) and possible additional amino acids from adonor immunoglobulin and a framework region from an accepting humanimmunoglobulin. Each humanized immunoglobulin chain usually comprises,in addition to the CDR's, amino acids from the donor immunoglobulinframework that are capable of interacting with the CDR's to effectbinding affinity, such as one or more amino acids that are immediatelyadjacent to a CDR in the donor immunoglobulin or those within about 3Aas predicted by molecular modeling. The heavy and light chains may eachbe designed by using any one, any combination, or all of the variousposition criteria described in U.S. Pat. Nos. 5,693,762; 5,693,761;5,585,089; and 5,530,101, each incorporated herein by reference. Whencombined into an intact antibody, the humanized immunoglobulins aresubstantially non-immunogenic in humans and retain substantially thesame affinity as the donor immunoglobulin to the original antigen.

[0330] An additional method for producing humanized antibodies isdescribed in U.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporatedherein by reference. This method combines the concept of humanizingantibodies with the phagemid libraries also described in detail herein.In a general sense, the method utilizes sequences from the antigenbinding site of an antibody or population of antibodies directed againstan antigen of interest. Thus for a single rodent antibody, sequencescomprising part of the antigen binding site of the antibody may becombined with diverse repertoires of sequences of human antibodies thatcan, in combination, create a complete antigen binding site.

[0331] The antigen binding sites created by this process differ fromthose created by CDR grafting, in that only the portion of sequence ofthe original rodent antibody is likely to make contacts with antigen ina similar maimer. The selected human sequences are likely to differ insequence and make alternative contacts with the antigen from those ofthe original binding site. However, the constraints imposed by bindingof the portion of original sequence to antigen and the shapes of theantigen and its antigen binding sites, are likely to drive the newcontacts of the human sequences to the same region or epitope of theantigen This process has therefore been termed “epitope imprintedselection” (EIS).

[0332] Starting with an animal antibody, one process results in theselection of antibodies that are partly human antibodies. Suchantibodies may be sufficiently similar in sequence to human antibodiesto be used directly in therapy or after alteration of a few keyresidues. Sequence differences between the rodent component of theselected antibody with human sequences could be minimized by replacingthose residues that differ with the residues of human sequences, forexample, by site directed mutagenesis of individual residues, or by CDRgrafting of entire loops. However, antibodies with entirely humansequences can also be created. EIS therefore offers a method for makingpartly human or entirely human antibodies that bind to the same epitopeas animal or partly human antibodies respectively. In EIS, repertoiresof antibody fragments can be displayed on the surface of filamentousphase and the genes encoding fragments with antigen binding activitiesselected by binding of the phage to antigen.

[0333] Additional methods for humanizing antibodies contemplated for usein the present invention are described in U.S. Pat. Nos. 5,750,078;5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864;4,935,496; and 4,816,567, each incorporated herein by reference.

[0334] E8. Mutagcncsis by PCR

[0335] Site-specific mutagenesis is a technique useful in thepreparation of individual antibodies through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, whether humanizing or not, by introducing oneor more nucleotide sequence changes into the DNA.

[0336] Although many methods are suitable for use in mutagenesis, theuse of the polymerase chain reaction (PCRT) is generally now preferred.This technology offers a quick and efficient method for introducingdesired mutations into a given DNA sequence. The following textparticularly describes the use of PCRT to introduce point mutations intoa sequence, as may be used to change the amino acid encoded by the givensequence. Adaptations of this method are also suitable for introducingrestriction enzyme sites into a DNA molecule.

[0337] In this method, synthetic oligonucleotides are designed toincorporate a point mutation at one end of an amplified segment.Following PCRT, the amplified fragments are blunt-ended by treating withKlenow fragments, and the blunt-ended fragments are then ligated andsubcloned into a vector to facilitate sequence analysis.

[0338] To prepare the template DNA that one desires to mutagenize, theDNA is subdloned into a high copy number vector, such as pUC19, usingrestriction sites flanking the area to be mutated. Template DNA is thenprepared using a plasmid miniprep. Appropriate oligonucleotide primersthat are based upon the parent sequence, but which contain the desiredpoint mutation and which are flanked at the 5′ end by a restrictionenzyme site, are synthesized using an automated synthesizer. It isgenerally required that the primer be homologous to the template DNA forabout 15 bases or so. Primers may be purified by denaturingpolyacrylamide gel electrophoresis, although this is not absolutelynecessary for use in PCRTM. The 5′ end of the oligonucleotides shouldthen be phosphorylated.

[0339] The template DNA should be amplified by PCR™, using theoligonucleotide primers that contain the desired point mutations. Theconcentration of MgCI₂ in the amplification buffer will generally beabout 15 mM. Generally about 20-25 cycles of PCR™ should be carried outas follows: denaturation, 35 sec. at 95° C.; hybridization, 2 min. at50° C.; and extension, 2 min. at 72° C. The PCR™ will generally includea last cycle extension of about 10 min. at 72° C. After the finalextension step, about 5 units of Klenow fragments should be added to thereaction mixture and incubated for a further 15 min. at about 30° C. Theexonuclease activity of the Klenow fragments is required to make theends flush and suitable for blunt-end cloning.

[0340] The resultant reaction mixture should generally be analyzed bynondenaturing agarose or acrylamide gel electrophoresis to verify thatthe amplification has yielded the predicted product. One would thenprocess the reaction mixture by removing most of the mineral oils,extracting with chloroform to remove the remaining oil, extracting withbuffered phenol and then concentrating by precipitation with 100%ethanol. Next, one should digest about half of the amplified fragmentswith a restriction enzyme that cuts at the flanking sequences used inthe oligonucleotides. The digested fragments are purified on a lowgelling/melting agarose gel.

[0341] To subelone the fragments and to check the point mutation, onewould subclone the two amplified fragments into an appropriatelydigested vector by blunt-end ligation. This would be used to transformE. coli, from which plasmid DNA could subsequently be prepared using aminiprep. The amplified portion of the plasmid DNA would then beanalyzed by DNA sequencing to confirm that the correct point mutationwas generated. This is important as Taq DNA polymerase can introduceadditional mutations into DNA fragments.

[0342] The introduction of a point mutation can also be effected usingsequential PCR™ steps. In this procedure, the two fragments encompassingthe mutation are annealed with each other and extended by mutuallyprimed synthesis. This fragment is then amplified by a second PCR™ step,thereby avoiding the blunt-end ligation required in the above protocol.In this method, the preparation of the template DNA, the generation ofthe oligonucleotide primers and the first PCR™ amplification areperformed as described above. In this process, however, the chosenoligonucleotides should be homologous to the template DNA for a stretchof between about 15 and about 20 bases and must also overlap with eachother by about 10 bases or more.

[0343] In the second PCR™ amplification, one would use each amplifiedfragment and each flanking sequence primer and carry PCR™ for betweenabout 20 and about 25 cycles, using the conditions as described above.One would again subclone the fragments and check that the point mutationwas correct by using the steps outlined above.

[0344] In using either of the foregoing methods, it is generallypreferred to introduce the mutation by amplifying as small a fragment aspossible. Of course, parameters such as the melting temperature of theoligonucleotide, as will generally be influenced by the GC content andthe length of the oligo, should also be carefully considered. Theexecution of these methods, and their optimization if necessary, will beknown to those of skill in the art, and are further described in variouspublications, such as Current Protocols in Molecular Biology, 1995incorporated herein by reference.

[0345] When performing site-specific mutagenesis, Table A can beemployed as a reference. TABLE A Amino Acids Codons Alanine Ala A GCAGCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAG GAU Glutamicacid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGGGGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA GAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0346] E9. Recombinant Expression and Delivery

[0347] Given that many methods are available for cloning antibodies,anti-aminophospholipid antibodies may be prepared by routine methods ofrecombinant expression. The term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript will generallybe translated into a protein. Thus, as intended herein, expressionpreferably includes both transcription of an anti-aminophospholipidantibody gene or DNA and translation of the mRNA into ananti-aminophospholipid antibody protein product.

[0348] For the expression of an anti-aminophospholipid antibody, once asuitable clone or clones have been obtained, whether they be cDNA basedor genomic, one may proceed to prepare an expression system or“construct” for recombinant antibody production. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system will beperformed by techniques generally known to those of skill in recombinantantibody expression (Sambrook et al., 1989; incorporated herein byreference). In order for the construct to effect expression of ananti-aminophospholipid antibody transcript, the polynucleotide encodingthe antibody will preferably be under the transcriptional control of apromoter that promotes expression in the chosen host cell.

[0349] Recombinantly produced antibodies may be purified and formulatedfor human administration. Alternatively, nucleic acids encodinganti-aminophospholipid antibodies may be delivered via gene therapy.Although naked recombinant DNA or plasmids may be employed, the use ofliposomes or vectors is preferred. The ability of certain viruses toenter cells via receptor-mediated endocytosis and to integrate into hostcell genome and express viral genes stably and efficiently have madethem attractive candidates for the transfer of foreign genes intomammalian cells. Preferred gene therapy vectors for use in the presentinvention will generally be viral vectors.

[0350] Retroviruses have promise as gene delivery vectors due to theirability to integrate their genes into the host genome, transferring alarge amount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines.Other viruses, such as adenovirLs, herpes simplex viruses (HSV),cytomegalo,irus (CMV), and adeno-associated virus (AAV), such as thosedescribed by U.S. Pat. No. 5,139,941 (incorporated herein by reference),may also be engineered to serve as vectors for gene transfer.

[0351] Although some viruses that can accept foreign genetic materialare limited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

[0352] In certain further embodiments, the gene therapy vector will beHSV. A factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (e.g., temporal, strength)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and can be grown to high titers.

[0353] Of course, in using viral delivery systems, one will desire topurify the virion sufficiently to render it essentially free ofundesirable contaminants, such as defective interfering viral particlesor endotoxins and other pyrogens such that it will not cause anyuntoward reactions in the cell, animal or individual receiving thevector construct. A preferred means of purifying the vector involves theuse of buoyant density gradients, such as cesium chloride gradientcentrifugation.

[0354] E10. Antibody Fragments

[0355] Irrespective of the source of the original anti-aminophospholipidantibody, either the intact antibody, antibody multimers, or any one ofa variety of functional, antigeni-bindinig regions of the antibody maybe used in the present invention. Exemplary functional regions includescFv, Fv, Fab′, Fab and F(ab′)₂ fragments of the anti-aminophospholipidantibodies. Techniques for preparing such constructs are well known tothose in the art and are further exemplified herein.

[0356] The choice of antibody construct may be influenced by variousfactors. For example, prolonged half-life can result from the activereadsorption of intact antibodies within the kidney, a property of theFc piece of immunoglobulin. IgG based antibodies, therefore, areexpected to exhibit slower blood clearance than their Fab′ counterparts.However, Fab′ fragment-based compositions will generally exhibit bettertissue penetrating capability.

[0357] Fab fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiol protease, papain. Papain mustfirst be activated by reducing the sulphydryl group in the active sitewith cysteine, 2-mercaptoethanol or dithiothreitol. Heavy met als in thestock enzyme should be removed by chelation with EDTA (2 mM) to ensuremaximum enzyme activity. Enzyme and substrate are normally mixedtogether in the ratio of 1:100 by weight. After incubation, the reactioncan be stopped by irreversible alkylation of the thiol group withiodoacetamide or simply by dialysis. The completeness of the digestionshould be monitored by SDS-PAGE and the various fractions separated byprotein A-Sepharose or ion exchange chromatography.

[0358] The usual procedure for preparation of F(ab′)₂ fragments from IgGof rabbit and human origin is limited proteolysis by the enzyme pepsin.The conditions, 100× antibody excess w/w in acetate buffer at pH 4.5,37° C., suggest that antibody is cleaved at the C-terminal side of theinter-heavy-chain disulfide bond. Rates of digestion of mouse IgG mayvary with subclass and it may be difficult to obtain high yields ofactive F(ab′)₂ fragments without some undigested or completely degradedIgG. In particular, IgG,, is highly susceptible to complete degradation.The other subclasses require different incubation conditions to produceoptimal results, all of which is known in the art.

[0359] Digestion of rat IgG by pepsin requires conditions includingdialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for fourhours with 1% w/w pepsin; IgG, and IgG₂. digestion is improved if firstdialyzed against 0.1 M formate buffer, pH 2.8, at 4° C., for 16 hoursfollowed by acetate buffer. IgG_(2b) gives more consistent results withincubation in staphylococcal V8 protease (3% w/w) in 0.1 M sodiumphosphate buffer, pH 7.8, for four hours at 37° C.

[0360] The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use offunctional, antigen-binding regions of antibodies, including scFv, Fv,Fab′, Fab and F(ab′)₂ fragments of the anti-aminophospholipidantibodies: U.S. Pat. Nos. 5,855,866 and 5,877,289; and U.S. applicationSer. Nos. 08/350,212 (U.S. Pat. No. 5,___,___ Issue Fee paid);08/482,369 (U.S. Pat. No. 5,___,___; Issue Fee paid); and 08/487,427(U.S. Pat. No. 5,___,___; Issue Fee paid).

[0361] F. Pharmaceutical Compositions

[0362] The most basic pharmaceutical compositions of the presentinvention will generally comprise an effective amount of at least afirst naked anti-aminophospholipid antibody, or antigen-binding fragmentthereof, dissolved or dispersed in a pharmaceutically acceptable carrieror aqueous medium. Combined therapeutics are also contemplated, and thesame type of underlying pharmaceutical compositions may be employed forboth single and combined medicaments.

[0363] The phrases “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards. Supplementary active ingredientscan also be incorporated into the compositions.

[0364] F1. Parenteral Formulations

[0365] The anti-aminophospholipid antibodies of the present inventionwill most often be formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, transdermal, or other such routes, including peristalticadministration and direct instillation into a tumor or disease site(intracavity administration). The preparation of an aqueous compositionthat contains an anti-aminophospholipid antibody as an active ingredientwill be known to those of skill in the art in light of the presentdisclosure. Typically, such compositions can be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forusing to prepare solutions or suspensions upon the addition of a liquidprior to injection can also be prepared; and the preparations can alsobe emulsified.

[0366] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions; formulations including sesameoil, peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. in all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

[0367] The anti-aminophospholipid antibody compositions can beformulated into a sterile aqueous composition in a neutral or salt form.Solutions of the anti-aminophospholipid antibodies as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

[0368] Suitable carriers include solvents and dispersion mediacontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants.

[0369] Under ordinary conditions of storage and use, all suchpreparations should contain a preservative to prevent the growth ofmicroorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0370] Prior to or upon formulation, the anti-aminophospholipidantibodies should be extensively dialyzed to remove undesired smallmolecular weight molecules, and/or lyophilized for more readyformulation into a desired vehicle, where appropriate. Sterileinjectable solutions are prepared by incorporating the activeanti-aminophospholipid antibodies in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as desired, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle that contains the basic dispersionmedium and the required other ingredients from those enumerated above.

[0371] In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation arevacuum-drying and freeze-drying techniques that yield a powder of theactive anti-arninophospholipid antibody ingredient, plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0372] Suitable pharmaceutical compositions in accordance with theinvention will generally include an amount of the anti-aminophospholipidantibody admixed with an acceptable pharmaceutical diluent or excipient,such as a sterile aqueous solution, to give a range of finalconcentrations, depending on the intended use. The techniques ofpreparation are generally well known in the art as exemplified byRemington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company,1980, incorporated herein by reference. It should be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

[0373] Upon formulation, anti-aminophospholipid antibody solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. Formulations are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets, pills, capsulesor other solids for oral administration, suppositories, pessaries, nasalsolutions or sprays, aerosols, inhalants, liposomal forms and the like.Pharmaceutical “slow release” capsules or compositions may also be used.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliveranti-aminophospholipid antibodies in accordance with the presentinvention.

[0374] F2. Liposomes and Nanocapsules

[0375] In certain embodiments, liposomes and/or nanoparticles may alsobe employed with the anti-aminophospholipid antibodies. The formationand use of liposomes is generally known to those of skill in the art, assummarized below.

[0376] Liposomes are formed from phospholipids that are dispersed in anaqueous medium and spontaneously form multilamellar concentric bilayervesicles (also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

[0377] Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

[0378] Liposomes interact with cells via four different mechanisms:Endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

[0379] Nanocapsules can generally entrap compounds in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

[0380] G. Therapeutic Kits

[0381] This invention also provides therapeutic kits comprisinganti-aminophospholipid antibodies for use in the present treatmentmethods. Such kits will generally contain, in suitable container means,a pharmaceutically acceptable formulation of at least oneanti-aminophospholipid antibody. The kits may also contain otherpharmaceutically acceptable formulations, either for diagnosis/imagingor combined therapy. For example, such kits may contain any one or moreof a range of chemotherapeutic or radiotherapeutic drugs;anti-angiogenic agents; anti-tumor cell antibodies; and/or anti-tumorvasculature or anti-tumor stroma immunotoxins or coaguligands.

[0382] The kits may have a single container (container means) thatcontains the anti-aminophospholipid antibody, with or without anyadditional components, or they may have distinct containers for eachdesired agent. Where combined therapeutics are provided, a singlesolution may be pre-mixed, either in a molar equivalent combination, orwith one component in excess of the other. Alternatively, each of theanti-aminophospholipid antibody and other anti-cancer agent componentsof the kit may be maintained separately within distinct containers priorto administration to a patient.

[0383] When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

[0384] Thle containers of the kit will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the anti-aminophospholipid antibody, and any other desired agent,may be placed and, preferably, suitably aliquoted. Where separatecomponents are included, the kit will also generally contain a secondvial or other container into which these are placed, enabling theadministration of separated designed doses. The kits may also comprise asecond/third container means for containing a sterile, pharmaceuticallyacceptable buffer or other diluent.

[0385] The kits may also contain a means by which to administer theanti-aminophospholipid antibody to an animal or patient, e.g, one ormore needles or syringes, or even an eye dropper, pipette, or other suchlike apparatus, from which the formulation may be injected into theanimal or applied to a diseased area of the body. The kits of thepresent invention will also typically include a means for containing thevials, or such like, and other component, in close confinement forcommercial sale, such as, e.g., injection or blow-molded plasticcontainers into which the desired vials and other apparatus are placedand retained.

[0386] H. Tumor Treatment

[0387] The most important use of the present invention is in thetreatment of vascularized, malignant tumors; with the treatment ofbenign tumors, such as BPH, also being contemplated. The invention mayalso be used in the therapy of other diseases and disorders having, as acomponent of the disease, prothrombotic blood vessels. Suchvasculature-associated diseases include diabetic retinopathy, maculardegeneration, vascular restenosis, including restenosis followingangioplasty, arteriovenous malformations (AVM), meningioma, hemangioma,neovascular glaucoma and psoriasis; and also angiofibroma, arthritis,rheumatoid arthritis, atherosclerotic plaques, corneal graftneovascularization, hemophilic joints, hypertrophic scars, osler-webersyndrome, pyogenic granuloma retrolental fibroplasia, scleroderma,trachoma, vascular adhesions, synovitis, dermatitis, various otherinflammatory diseases and disorders, and even endometriosis.

[0388] The anti-aminophospholipid antibody treatment of the invention ismost preferably exploited for the treatment of solid tumors. Such usesmay employ anti-aminophospholipid antibodies alone or in combinationwith chemotherapeutic, radiotherapeutic, apoptopic, anti-angiogenicagents and/or immunotoxins or coaguligands. The anti-aminophospholipidantibody methods provided by this invention are broadly applicable tothe treatment of any malignant tumor having a vascular component.Typical vascularized tumors are the solid tumors, particularlycarcinomas, which require a vascular component for the provision ofoxygen and nutrients. Exemplary solid tumors that may be treated usingthe invention include, but are not limited to, carcinomas of the lung,breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, and the like.

[0389] The present invention is contemplated for use in the treatment ofany patient that presents with a solid tumor. However, in that thisinvention is particularly successful in the treatment of solid tumors ofmoderate or large sizes, patients in these categories are likely toreceive more significant benefits from treatment in accordance with themethods and compositions provided herein.

[0390] Therefore, in general, the invention can be used to treat tumorsof about 0.3-0.5 cm and upwards, although it is a better use of theinvention to treat tumors of greater than 0.5 cm in size. From thestudies already conducted in acceptable animal models, it is believedthat patients presenting with tumors of between about 1.0 and about 2.0cm in size will be in the preferred treatment group of patients foranti-aminophospholipid antibody therapy, although tumors up to andincluding the largest tumors found in humans may also be treated.

[0391] Although the present invention is not generally intended as apreventative or prophylactic treatment, use of the invention iscertainly not confined to the treatment of patients having tumors ofonly moderate or large sizes. There are many reasons underlying thisaspect of the breadth of the invention. For example, a patientpresenting with a primary tumor of moderate size or above may also havevarious other metastatic tumors that are considered to be small-sized oreven in the earlier stages of metastatic tumor seeding. Given that theanti-aminophospholipid antibodies, or combinations, of the invention aregenerally administered into the systemic circulation of a patient, theywill naturally have effects on the secondary, smaller and metastatictumors, although this may not be the primary intent of the treatment.Furthermore, even in situations where the tumor mass as a whole is asingle small tumor, certain beneficial anti-tumor effects will resultfrom the use of the present anti-aminophospholipid antibody treatment.

[0392] The guidance provided herein regarding the most suitable patientsfor use in connection with the present invention is intended as teachingthat certain patient's profiles may assist with the selection ofpatients for treatment by the present invention. The pre-selection ofcertain patients, or categories of patients, does not in any way negatethe basic usefulness of the present invention in connection with thetreatment of all patients having a vascularized tumor. A furtherconsideration is the fact that the assault on the tumor provided by theanti-aminophospholipid antibody therapy of the invention may predisposethe tumor to further therapeutic treatment, such that the subsequenttreatment results in an overall synergistic effect or even leads tototal remission or cure.

[0393] It is not believed that any particular type of tumor should beexcluded from treatment using the present invention. However, the typeof tumor cells may be relevant to the use of the invention incombination with secondary therapeutic agents, particularlychemotherapeutics and anti-tumor cell immunotoxins. As the effect of thepresent therapy is to destroy the tumor vasculature, and as thevasculature is substantially or entirely the same in all solid tumors,it will be understood that the present anti-aminophospholipidmethodology is widely or entirely applicable to the treatment of allsolid tumors, irrespective of the particular phenotype or genotype ofthe tumor cells themselves. The data presented herein is compelling asit shows impressive results in a tumor model that is resistant tonecrosis.

[0394] Therapeutically effective closes of anti-aminophosplholipidantibodies are readily determinable using data from an animal model, asshown in the studies detailed herein. Experimental animals bearing solidtumors are frequently used to optimize appropriate therapeutic dosesprior to translating to a clinical environment. Such models are known tobe very reliable in predicting effective anti-cancer strategies. Forexample, mice bearing solid tumors, such as used in the Examples, arewidely used in pre-clinical testing. The inventors have used suchart-accepted mouse models to determine working ranges of nakedanti-aminophospholipid antibodies that give beneficial anti-tumoreffects with minimal toxicity.

[0395] As is known in the art, there are realistic objectives that maybe used as a guideline in connection with pre-clinical testing beforeproceeding to clinical treatment. However, due to the safety alreadydemonstrated in accepted models, pre-clinical testing of the presentinvention will be more a matter of optimization, rather than to confirmeffectiveness. Thus, pre-clinical testing may be employed to select themost advantageous anti-aminophospholipid antibodies, doses orcombinations.

[0396] Any anti-aminophospholipid antibody dose, or combined medicament,that results in any consistent detectable tumor vasculature destruction,thrombosis and anti-tumor effects will still define a useful invention.Destructive, thrombotic and necrotic effects should be observed inbetween about 10% and about 40-50% of the tumor blood vessels and tumortissues, upwards to between about 50% and about 99% of such effectsbeing observed. The present invention may also be effective againstvessels downstream of the tumor, i.e., target at least a sub-set of thedraining vessels, particularly as cytokines released from the tumor willbe acting on these vessels, changing their antigenic profile.

[0397] It will also be understood that even in such circumstances wherethe anti-tumor effects of the anti-aminophospholipid antibody dose, orcombined therapy, are towards the low end of this range, it may be thatthis therapy is still equally or even more effective than all otherknown therapies in the context of the particular tumor targets. It isunfortunately evident to a clinician that certain tumors cannot beeffectively treated in the intermediate or long term, but that does notnegate the usefulness of the present therapy, particularly where it isat least about as effective as the other strategies generally proposed.

[0398] In designing appropriate doses of anti-aminophospholipidantibodies, or combined therapeutics, for the treatment of vascularizedtumors, one may readily extrapolate from the animal studies describedherein in order to arrive at appropriate doses for clinicaladministration. To achieve this conversion, one would account for themass of the agents administered per unit mass of the experimental animaland, preferably, account for the differences in the body surface areabetween the experimental animal and the human patient. All suchcalculations are well known and routine to those of ordinary skill inthe art.

[0399] For example, in taking the successful dose of 20 μg antibody permouse (total body weight of about 20 g), and applying standardcalculations based upon mass and surface area, effective doses for usein human patients would be between about 1 mg and about 500 mgs antibodyper patient, and preferably, between about 10 mgs and about 100 mgsantibody per patient. In addition to all variable clinical andtherapeutic parameters, this variation also accounts for the presenttumor necrosis data being generated using a tumor model that isresistant to necrosis, and in which less tumor vessel staining wasobserved than in other models.

[0400] Accordingly, using this information, the inventors contemplatethat useful low doses of naked anti-aminophospholipid antibodies forhuman administration will be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or about 30 mgs or so per patient; and useful high doses of nakedanti-aminophospholipid antibodies for human administration will be about250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or about 500 mgs or soper patient. Useful intermediate doses of naked anti-aminophospholipidantibodies for human administration are contemplated to be about 35, 40,50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or about 225 mgs or so perpatient. Any particular range using any of the foregoing recitedexemplary doses or any value intermediate between the particular statedranges is also contemplated.

[0401] In general, dosage ranges of between about 5-100 mgs, about 10-80nmgs, about 20-70 mgs, about 25-60 mgs, or about 30-50 mgs or so ofantibody per patient will be preferred. Notwithstanding these statedranges, it will be understood that, given the parameters and detailedguidance presented herein, further variations in the active or optimalranges will be encompassed within the present invention. Although dosesin and around about 5 or 10 to about 70, 80, 90 or 100 mgs per patientare currently preferred, it will be understood that lower doses may bemore appropriate in combination with other agents, and that high dosescan still be tolerated, particularly given the enhanced safety of theunconjugated anti-aminophospholipid antibodies for use in the invention.The use of human or humanized naked anti-aminophospholipid antibodiesrenders the present invention even safer for clinical use, furtherreducing the chances of significant toxicity or side effects in healthytissues.

[0402] The intention of the therapeutic regimens of the presentinvention is generally to produce significant anti-tumor effects whilststill keeping the dose below the levels associated with unacceptabletoxicity. In addition to varying the dose itself, the administrationregimen can also be adapted to optimize the treatment strategy. Acurrently preferred treatment strategy is to administer between about1-500 mgs, and preferably, between about 10-100 mgs of theanti-aminophospholipid antibody, or therapeutic cocktail containingsuch, about 3 times within about a 7 day period. For example, doseswould be given on about day 1, day 3 or 4 and day 6 or 7.

[0403] In administering the particular doses themselves, one wouldpreferably provide a pharmaceutically acceptable composition (accordingto FDA standards of sterility, pyrogenicity, purity and general safety)to the patient systemically. Intravenous injection is generallypreferred, and the most preferred method is to employ a continuousinfusion over a time period of about 1 or 2 hours or so. Although it isnot required to determine such parameters prior to treatment using thepresent invention, it should be noted that the studies detailed hereinresult in at least some thrombosis being observed specifically in theblood vessels of a solid tumor within about 12-24 hours of injection,and that the tumor cells themselves begin to die within about 24 to 72hours. Widespread tumor necrosis is generally observed in the next about48-96 hours, up to and including greater than 60% necrosis beingobserved.

[0404] Naturally, before wide-spread use, clinical trials will beconducted. The various elements of conducting a clinical trial,including patient treatment and monitoring, will be known to those ofskill in the art in light of the present disclosure. The followinginformation is being presented as a general guideline for use inestablishing such trials.

[0405] Patients chosen for the first anti-aminophospholipid antibodytreatment studies will have failed to respond to at least one course ofconventional therapy, and will have objectively measurable disease asdetermined by physical examination, laboratory techniques, and/orradiographic procedures. Any chemotherapy should be stopped at least 2weeks before entry into the study. Where murine monoclonal antibodies orantibody portions are employed, the patients should have no history ofallergy to mouse immunoglobulin.

[0406] Certain advantages will be found in the use of an indwellingcentral venous catheter with a triple lumen port. Theanti-aminophospholipid antibodies should be filtered, for example, usinga 0.22μ filter, and diluted appropriately, such as with saline, to afinal volume of 100 ml. Before use, the test sample should also befiltered in a similar manner, and its concentration assessed before andafter filtration by determining the A₂₈₀. The expected recovery shouldbe within the range of 87% to 99%, and adjustments for protein loss canthen be accounted for.

[0407] These anti-aminophospholipid antibodies may be administered overa period of approximately 4-24 hours, with each patient receiving 2-4infusions at 2-7 day intervals. Administration can also be performed bya steady rate of infusion over a 7 day period. The infusion given at anydose level should be dependent upon any toxicity observed. Hence, ifGrade II toxicity was reached after any single infusion, or at aparticular period of time for a steady rate infusion, further dosesshould be withheld or the steady rate infusion stopped unless toxicityimproved. Increasing doses of anti-aminophospholipid antibodies shouldbe administered to groups of patients until approximately 60% ofpatients showed unacceptable Grade III or IV toxicity in any category.Doses that are ⅔ of this value are defined as the safe dose.

[0408] Physical examination, tumor measurements, and laboratory testsshould, of course, be performed before treatment and at intervals up to1 month later. Laboratory tests should include complete blood counts,serum creatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the administered anti-aminophospholipid antibodies, andantibodies against any portions thereof. Immunological analyses of sera,using any standard assay such as, for example, an ELISA or RIA, willallow the pharmacokinetics and clearance of the anti-aminophospholipidtherapeutic agent to be evaluated.

[0409] To evaluate the anti-tumor responses, the patients should beexamined at 48 hours to 1 week and again at 30 days after the lastinfusion. When palpable disease was present, two perpendicular diametersof all masses should be measured daily during treatment, within 1 weekafter completion of therapy, and at 30 days. To measure nonpalpabledisease, serial CT scans could be performed at 1-cm intervalstlhroughout the chest, abdomen, and pelvis at 48 hours to 1 week andagain at 30 days. Tissue samples should also be evaluatedhistologically, and/or by flow cytometry, using biopsies from thedisease sites or even blood or fluid samples if appropriate.

[0410] Clinical responses may be defined by acceptable measure. Forexample, a complete response may be defined by the disappearance of allmeasurable tumor 1 month after treatment. Whereas a partial response maybe defined by a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

[0411] In light of results from clinical trials, such as those describedabove, an even more precise treatment regimen may be formulated. Evenso, some variation in dosage may later be necessary depending on thecondition of the subject being treated. The physician responsible foradministration will, in light of the present disclosure, be able todetermine the appropriate dose for the individual subject. Suchoptimization and adjustment is routinely carried out in the art and byno means reflects an undue amount of experimentation.

[0412] I. Tumor Imaging

[0413] The present invention further provides combined tumor treatmentand imaging methods, based upon anti-aminophospholipid binding ligands.Anti-aminophospholipid binding proteins or antibodies that are linked toone or more detectable agents are envisioned for use in pre-imaging thetumor, forming a reliable image prior to the treatment, which itselftargets the aminophospholipid markers. In addition to antibodies, theuse of detectably labeled annexins and other aminophospholipid bindingligands is contemplated, as disclosed and claimed in first and secondprovisional applications Ser. Nos. 60/092,589 (filed Jul. 13, 1998) and60/110,600 (filed Dec. 02, 1998) and in co-filed U.S. and PCT patentapplications (Attorney Docket Nos. 4001.002300, 4001.002382, 4001.002383and 4001.002210), each specifically incorporated herein by reference.

[0414] The anti-aminophospholipid imaging ligands or antibodies, orconjugates thereof, will generally comprise an anti-aminophospholipidantibody or binding ligand operatively attached, or conjugated to, adetectable label. “Detectable labels” are compounds or elements that canbe detected due to their specific functional properties, or chemicalcharacteristics, the use of which allows the component to which they areattached to be detected, and further quantified if desired. Preferably,the detectable labels are those detectable in vivo using non-invasivemethods.

[0415] Antibody and binding protein conjugates for use as diagnosticagents generally fall into two classes, those for use in in vitrodiagnostics, such as in a variety of immunoassays, and those for use invivo diagnostic protocols. It is the in vivo imaging methods that areparticularly intended for use with this invention.

[0416] Many appropriate imaging agents are known in the art, as aremethods for their attachment to antibodies and binding ligands (see, eg,U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein byreference). Certain attachment methods involve the use of a met alchelate complex employing, for example, an organic chelating agent sucha DTPA attached to the antibody (U.S. Pat. No. 4,472,509). Monoclonalantibodies may also be reacted with an enzyme in the presence of acoupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate.

[0417] An example of detectable labels are the paramagnetic ions. Inthis case, suitable ions include chromium (III), manganese (II), iron(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III), withgadolinium being particularly preferred.

[0418] Ions useful in other contexts, such as X-ray imaging, include butare not limited to lanthanum (III), gold (III), lead (II), andespecially bismuth (III). Fluorescent labels include rhodamine,fluorescein and renographin. Rhodamine and fluorescein are often linkedvia an isothiocyanate intermediate.

[0419] In the case of radioactive isotopes for diagnostic applications,suitable examples include ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt,⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodineodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, ⁷⁵selenium,³⁵sulphur, technetium^(99m) and yttrium⁹⁰. ¹²⁵I is often being preferredfor use in certain embodiments, and technicium^(99m) and indium¹¹¹ arealso often preferred due to their low energy and suitability for longrange detection.

[0420] Radioactively labeled anti-aminophospholipid antibodies andbinding ligands for use in the present invention may be producedaccording to well-known methods in the art. For instance, intermediaryfunctional groups that are often used to bind radioisotopic met allicions to antibodies are diethylenetriaminepentaacetic acid (DTPA) andethylene diaminetetracetic acid (EDTA).

[0421] Monoclonal antibodies can also be iodinated by contact withsodium or potassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Anti-aminophospholipid antibodies according to the invention may belabeled with technetium-⁹⁹m by ligand exchange process, for example, byreducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex colum and applying the antibody to thiscolum; or by direct labeling techniques, e.g., by incubatingpertechnate, a reducing agent such as SNCl₂, a buffer solution such assodium-potassium phthalate solution, and the antibody.

[0422] Any of the foregoing type of detectably labeledanti-aminophospholipid antibodies and aminophospholipid binding ligandsmay be used in the imaging aspects of the present invention. Althoughnot previously proposed for use in combined tumor imaging and treatment,the detectably-labeled annexins of U.S. Pat. No. 5,627,036; WO 95/19791;WO 95/27903; WO 95/34315; WO 96/17618; and WO 98/04294; eachincorporated herein by reference; may also be employed.

[0423] WO 95/27903 (incorporated herein by reference) provides annexinsfor use in detecting apoptotic cells. Any of the annexin-detectableagent markers of WO 95/27903 may be used herein, although it will beknown that certain of these are more suitable for in vitro uses. WO95/27903 is also specifically incorporated herein by reference forpurposes of providing detectable kits that may be adapted for combineduse with the therapeutics of the present invention.

[0424] Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO 98/04294;are also incorporated herein by reference for purposes of furtherdescribing radiolabelled annexin conjugates for diagnostic imaging. Theintent of each of the foregoing documents is to provide radiolabelledannexins for use in imaging vascular thromboses, particularly in or nearthe heart, such as in deep vein thrombosis, pulmonary embolism,myocardial infarction, atrial fibrillation, problems with prostheticcardiovascular materials, stroke, and the like. These radiolabelledannexins were also proposed for use in imaging activated platelets,e.g., in conditions such as abscesses, restenosis, inflammationofjoints, clots in cerebral arteries, etc.

[0425] U.S. Pat. No. 5,627,036 (incorporated herein by reference) alsogenerally concerns ‘annexine’ (annexin) binding ligands for use inanalyzing platelet phosphatidylserine. It is explained in U.S. Pat. No.5,627,036 that hemostatic disorders, such as arterial, coronary andvenous thrombosis, are usually idiopathic, which makes prediction andprevention difficult. To recognize such hemostatic disorders earlier,the detection of activated platelets is proposed. The detectably labeledannexins compositions are thus disclosed in order to detect activatedplatelets in hemostatic disorders (U.S. Pat. No. 5,627,036).

[0426] Although proposing a wide range of diagnostic uses, none of WO95/19791; WO 95/34315; WO 96/17618; or WO 98/04294 make reference toimaging the vasculature of solid tumors. Neither does U.S. Pat. No.5,627,036 make any such suggestions. Nonetheless, the discloseddetectable and radiolabelled annexin compositions per se may now be usedto advantage in this regard, in light of the surprising discoveriesdisclosed herein.

[0427] In particular, U.S. Pat. No. 5,627,036 (incorporated herein byreference) discloses annexins detectably labeled with fluoresceinisothiocyanate; radioisotopes of halogens, technetium, lead, mercury,thallium or indium; and paramagnetic contrast agents.

[0428] WO 95/19791 (incorporated herein by reference) providesconjugates of annexin bonded to an N₂S₂ chelate that can beradiolabelled by complexing a radionuclide to the chelate. WO 95/34315(incorporated herein by reference) provides annexin conjugatescomprising one or more galactose residues with the N₂S₂ chelate. Thegalactose moiety is said to facilitate the rapid elimination of theradiolabelled conjugate from the circulation, reducing radiation damageto non-target tissues and background ‘noise.’

[0429] WO 96/17618 (incorporated herein by reference) in turn providesannexin conjugates suitable for radiolabeling with diagnostic imagingagents that comprise an annexin with a cluster of galactose residues andan N₂S₂ chelate. These are reported to have a shorter circulatinghalf-life and a higher binding affinity for target sites than theforegoing radiolabeled annexin-galactose conjugates.

[0430] Still further radiolabeled annexin conjugates are provided by WO98/04294 (incorporated herein by reference). These conjugates comprisean annexin that is modified to provide an accessible sulphydryl groupconjugated to a hexose moiety that is recognized by a mammalian liverreceptor. Annexin multimer conjugates and chelating compounds conjugatedvia esterase-sensitive bonds are also provided.

[0431] Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO 98/04294;are also specifically incorporated herein by reference for purposes ofproviding annexin conjugate components for radiolabelling that areamenable to packaging in “cold kits”, i. e., wherein the components areprovided in separate vials. U.S. Pat. No. 5,627,036 similarly provideskits comprising a carrier being compartmentalized to receive detectablylabeled annexins that may be adapted for use herewith.

[0432] Although suitable for use in in vitro diagnostics, the presentaminophospholipid detection methods are more intended for forming animage of the tumor vasculature of a patient prior to treatment withtherapeutic agent-targeting agent constructs. The in vivo diagnostic orimaging methods generally comprise administering to a patient adiagnostically effective amount of an anti-aminophospholipid antibody orbinding ligand that is conjugated to a marker that is detectable bynon-invasive methods. The antibody- or binding ligand-marker conjugateis allowed sufficient time to localize and bind to the aminophospholipidexpressed on the luminal surface of the tumor vasculature. The patientis then exposed to a detection device to identify the detectable marker,thus forming an image of the tumor vasculature.

[0433] The nuclear magnetic spin-resonaiice isotopes, such asgadolinium, are detected using a nuclear magnetic imaging device; andradioactive substances, such as technicium^(99m) or indium¹¹¹, aredetected using a gamma scintillation camera or detector. U.S. Pat. No.5,627,036 is also specifically incorporated herein by reference forpurposes of providing even further guidance regarding the safe andeffective introduction of such detectably labeled constructs into theblood of an individual, and means for determining the distribution ofthe detectably labeled annexin extracorporally, e.g., using a gammascintillation camera or by magnetic resonance measurement.

[0434] Dosages for imaging embodiments are generally less than fortherapy, but are also dependent upon the age and weight of a patient. Aone time dose of between about 0.1, 0.5 or about 1 mg and about 9 or 10mgs, and more preferably, of between about 1 mg and about 5-10 mgs ofanti-aminophospholipid antibody- or aminophospholipid bindingligand-conjugate per patient is contemplated to be useful. U.S. Pat. No.5,627,036; and WO 95/19791, each incorporated herein by reference, arealso instructive regarding doses of detectably-labeled annexins.

[0435] J. Combination Therapies

[0436] The anti-aminophospholipid antibody treatment methods of thepresent invention may be combined with any other methods generallyemployed in the treatment of the particular tumor, disease or disorderthat the patient exhibits. So long as a particular therapeutic approachis not known to be detrimental to the patient's condition in itself, anddoes not significantly counteract the anti-aminophospholipid antibodytreatment, its combination with the present invention is contemplated.

[0437] In connection solid tumor treatment, the present invention may beused in combination with classical approaches, such as surgery,radiotherapy, chemotherapy, and the like. The invention thereforeprovides combined therapies in which anti-aminophospholipid antibodiesare used simultaneously with, before, or after surgery or radiationtreatment; or are administered to patients with, before, or afterconventional chemotherapeutic, radiotherapeutic or anti-angiogenicagents, or targeted immunotoxins or coaguligands.

[0438] Combination therapy for other vascular diseases is alsocontemplated. A particular example of such is benign prostatichyperplasia (BPH), which may be treated with anti-aminophospholipidantibodies in combination other treatments currently practiced in theart. For example, targeting of immunotoxins to markers localized withinBPI-I, such as PSA.

[0439] When one or more agents are used in combination with theanti-aminophospholipid antibody therapy, there is no requirement for thecombined results to be additive of the effects observed when eachtreatment is conducted separately. Although at least additive effectsare generally desirable, any increased anti-tumor effect above one ofthe single therapies would be of benefit. Also, there is no particularrequirement for the combined treatment to exhibit synergistic effects,although this is certainly possible and advantageous. Agentsparticularly contemplated for use in achieving potentially synergisticeffects are those that injure, or induce apoptosis in, the tumorendothelium, as such injury or apoptosis should amplify the overalltherapeutic effect.

[0440] To practice combined anti-tumor therapy, one would simplyadminister to an animal an anti-aminophospholipid antibody incombination with another anti-cancer agent in a mariner effective toresult in their combined anti-tumor actions within the animal. Theagents would therefore be provided in amounts effective and for periodsof time effective to result in their combined presence within the tumorvasculature and their combined actions in the tumor environment. Toachieve this goal, the anti-aminophospholipid antibodies and anti-canceragents may be administered to the animal simultaneously, either in asingle composition, or as two distinct compositions using differentadministration routes.

[0441] Alternatively, the anti-aminophospholipid antibody treatment mayprecede, or follow, the anti-cancer agent treatment by, e.g., intervalsranging from minutes to weeks. In certain embodiments where theanti-cancer agent and anti-aminophospholipid antibody are appliedseparately to the animal, one would ensure that a significant period oftime did not expire between the time of each delivery, such that theanti-cancer agent and anti-aminophospholipid antibody composition wouldstill be able to exert an advantageously combined effect on the tumor.In such instances, it is contemplated that one would contact the tumorwith both agents within about 5 minutes to about one week of each otherand, more prcferably, within about 12-72 hours of each other, with adelay time of only about 12-48 hours being most preferred.

[0442] Exemplary anti-cancer agents that would be given prior to theanti-aminophospholipid antibody are agents that induce the expression ofaminophospholipids within the tumor vasculature. For example, agentsthat stimulate localized calcium production and/or that induce apoptosiswill generally result in increased PS expression, which can then betargeted using a subsequent anti-PS antibody. Anti-aminophospholipidantibodies would be first administered in other situations to causetumor destruction, followed by anti-angiogenic therapies or therapiesdirected to targeting necrotic tumor cells.

[0443] The general use of combinations of substances in cancer treatmentis well know. For example, U.S. Pat. No. 5,710,134 (incorporated hereinby reference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxic“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

[0444] In some situations, it may even be desirable to extend the timeperiod for treatment significantly, where several days (2, 3, 4, 5, 6 or7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1,2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.This would be advantageous in circumstances where one treatment wasintended to substantially destroy the tumor, such as theanti-aminophospholipid antibody treatment, and another treatment wasintended to prevent micrometastasis or tumor re-growth, such as theadministration of an anti-angiogenic agent. The EN 7/44 antibody ofHagemeier et al. (1986) is not believed to be an effectiveanti-angiogenic agent, lacking binding to a surface accessible antigen,amongst other deficiencies.

[0445] It also is envisioned that more than one administration of eitherthe anti-aminophospholipid antibody or the anti-cancer agent will beutilized. The anti-aminophospholipid antibodies and anti-cancer agentsmay be administered interchangeably, on alternate days or weeks; or asequence of anti-aminophospholipid antibody treatment may be given,followed by a sequence of anti-cancer agent therapy. In any event, toachieve tumor regression using a combined therapy, all that is requiredis to deliver both agents in a combined amount effective to exert ananti-tumor effect, irrespective of the times for administration.

[0446] In terms of surgery, any surgical intervention may be practicedin combination with the present invention. In connection withradiotherapy, any mechanism for inducing DNA damage locally within tumorcells is contemplated, such as γ-irradiation, X-rays, UV-irradiation,microwaves and even electronic emissions and the like. The directeddelivery of radioisotopes to tumor cells is also contemplated, and thismay be used in connection with a targeting antibody or other targetingmeans.

[0447] Cytokine therapy also has proven to be an effective partner forcombined therapeutic regimens. Various cytokines may be employed in suchcombined approaches. Examples of cytokines include IL-1a IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,TGF-β, GM-CSF, M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG,MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administeredaccording to standard regimens, consistent with clinical indicationssuch as the condition of the patient and relative toxicity of thecytokine. Uteroglobins may also be used to prevent or inhibit metastases(U.S. Pat. No. 5,696,092; incorporated herein by reference).

[0448] J1. Chemotherapeutics

[0449] In certain embodiments, the anti-aminophospholipid antibodies ofthe present invention may be administered in combination with achemotherapeutic agent. Chemotherapeutic drugs can kill proliferatingtumor cells, enhancing the necrotic areas created by the overalltreatment. The drugs can thus enhance the thrombotic action of theanti-aminophospholipid antibodies.

[0450] By inducing the formation of thrombi in tumor vessels, theanti-aminophospholipid antibodies can enhance the action of thechemotherapeutics by retaining or trapping the drugs within the tumor.The chemotherapeutics are thus retained within the tumor, while the restof the drug is cleared from the body. Tumor cells are thus exposed to ahigher concentration of drug for a longer period of time. Thisentrapment of drug within the tumor makes it possible to reduce the doseof drug, making the treatment safer as well as more effective.

[0451] Irrespective of the underlying mechanism(s), a variety ofchemotherapeutic agents may be used in the combined treatment methodsdisclosed herein. Chemotherapeutic agents contemplated as exemplaryinclude, e.g, tamoxifen, taxol, vincristine, vinblastine, etoposide(VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D,mitomycin C, cisplatin (CDDP), combretastatin(s) and derivatives andprodrugs thereof.

[0452] As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

[0453] Further useful agents include compounds that interfere with DNAreplication, mitosis, chromosomal segregation and/or tubulin activity.Such chemotherapeutic compounds include adriamycin, also known asdoxorubicin, etoposide, verapamil, podophyllotoxin(s), combretastatin(s)and the like. Widely used in a clinical setting for the treatment ofneoplasms, these compounds are administered through bolus injectionsintravenously at doses ranging from 25-75 mg/m² at 21 day intervals foradriamycin, to 35-50 mg/m² for etoposide intravenously or double theintravenous dose orally.

[0454] Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorotiracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

[0455] Exemplary chemotherapeutic agents that are useful in connectionwith combined therapy are listed in Table B. Each of the agents listedtherein are exemplary and by no means limiting. The skilled artisan isdirected to “Remington's Pharmaceutical Sciences” 15th Edition, chapter33, in particular pages 624-652. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The physician responsible for administration will be able todetermine the appropriate dose for the individual subject. TABLE BCHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC DISEASE NONPROPRIETARYNAMES CLASS TYPE OF AGENT (OTHER NAMES) DISEASE Alkylating NitrogenMechlorethamine (HN₂) Hodgkin's disease, non-Hodgkin's Agents Mustardslymphomas Cyclophosphamide Acute and chronic lymphocytic Ifosfamideleukemias, Hodgkin's disease, non Hodgkin's lymphomas, multiple myeloma,neuroblastoma, breast, ovary, lung, Wilms' tumor, cervix, testis,soft-tissue sarcomas Melphalan (L-sarcolysin) Multiple myeloma, breast,ovary Chlorambucil Chronic lymphocytic leukemia, primarymacroglobulinemia, Hodgkin's disease, non-Hodgkin's lymphomasEthylenimenes and Hexamethylmelamine Ovary Methylmelamines ThiotepaBladder, breast, ovary Alkyl Sulfonates Busulfan Chronic granulocyticleukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, multiple myeloma, malignant melanomaLomustine (CCNU) Hodgkin's disease, non-Hodgkin's lymphomas, primarybrain tumors, small-cell lung Semustine (methyl-CCNU) Primary braintumors, stomach, colon Streptozocin Malignant pancreatic insulinoma,(streptozotocin) malignant carcinoid Triazines Dacarbazine (DTIC;Malignant melanoma, Hodgkin's dimethyltriazenoimidaz disease,soft-tissue sarcomas olecarboxamide) Antimetab- Folic Acid MethotrexateAcute lymphocytic leukemia, olites Analogs (amethopterin)choriocarcinoma, mycosis fungoides, breast, head and neck, lung,osteogenic sarcoma Pyrimidine Analogs Fluouracil (5-fluorouracil;Breast, colon, stomach, pancreas, 5-FU) ovary, head and neck, urinarybladder, Floxuridine (fluorode- premalignant skin lesions (topical)oxyuridine; FUdR) Cytarabine (cytosine Acute granulocytic and acutearabinoside) lymphocytic leukemias Purine Analogs and MercaptopurineAcute lymphocytic, acute Related Inhibitors (6-mercaptopurine;granulocytic and chronic granulocytic 6-MP) leukemias Thioguanine Acutegranulocytic, acute (6-thioguanine; TG) lymphocytic and chronicgranulocytic leukemias Pentostatin Hairy cell leukemia, mycosis(2-deoxycoformycin) fungoides, chronic lymphocytic leukemia NaturalVinca Alkaloids Vinblastine (VLB) Hodgkin's disease, non-Hodgkin'sProducts lymphomas, breast, testis Acute lymphocytic leukemia,Vincristine neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin'sdisease, non-Hodgkin's lymphomas, small-cell lung EpipodophyllotoxinsEtoposide Testis, small-cell lung and other lung, Tertiposide breast,Hodgkin's disease, non- Hodgkin's lymphomas, acute granulocyticleukemia, Kaposi's sarcoma Antibiotics Dactinomycin Choriocarcinoma,Wilms' tumor, (actinomycin D) rhabdomyosarcoma, testis, Kaposi's sarcomaDaunorubicin Acute granulocytic and acute (daunomycin; lymphocyticleukemias rubidomycin) Doxorubicin Soft-tissue, osteogenic and othersarcomas; Hodgkin's disease, non- Hodgkin's lymphomas, acute leukemias,breast, genitourinary, thyroid, lung, stomach, neuroblastoma BleomycinTestis, head and neck, skin, esophagus, lung and genitourinary tract;Hodgkin's disease, non- Hodgkin's lymphomas Plicamycin (mithramycin)Testis, malignant hypercalcemia Mitomycin (mitomycin C) Stomach, cervix,colon, breast, pancreas, bladder, head and neck Enzymes L-AsparaginaseAcute lymphocytic leukemia Biological Response Interferon alfa Hairycell leukemia., Kaposi's Modifiers sarcoma, melanoma, carcinoid, renalcell, ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides,multiple myeloma, chronic granulocytic leukemia Testis, ovary, bladder,head and neck, Miscellaneous Platinum Coordination Cisplatin (cis-DDP)lung, thyroid, cervix, endometrium, Agents Complexes Carboplatinneuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acutegranulocytic leukemia, breast Substituted Urea Hydroxyurea Chronicgranulocytic leukemia, polycythemia vera, essental thrombocytosis,malignant melanoma Methyl Hydrazine Procarbazine Hodgkin's diseaseDerivative (N-methylhydrazine, MIH) Adrenocortical Mitotane (o,p'-DDD)Adrenal cortex Suppressant Aminoglutethimide Breast Adrenocortico-Prednisone (several other Acute and chronic lymphocytic steroidsequivalent leukemias, non-Hodgkin's lymphomas, preparations available)Hodgkin's disease, breast Hormones and Progestins HydroxyprogesteroneAntagonists caproate Medroxyprogesterone Endometrium, breast acetateMegestrol acetate Estrogens Diethylstilbestrol Ethinyl estradiol (otherBreast, prostate preparations available) Antiestrogen Tamoxifen BreastAndrogens Testosterone propionate Breast Fluoxymesterone (otherpreparations available) Antiandrogen Flutamide ProstateGonadotropin-releasing Leuprolide Prostate hormone analog

[0456] J2. Anti-Angiogenics

[0457] The term “angiogenesis” refers to the generation of new bloodvessels, generally into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fet al and embryonic development and formation of thecorpus luteum, endometrium and placenta. Uncontrolled (persistent and/orunregulated) angiogenesis is related to various disease states, andoccurs during tumor development and metastasis.

[0458] Both controlled and uncontrolled angiogenesis are thought toproceed in a similar manner. Endothelial cells and pericytes, surroundedby a basement membrane, form capillary blood vessels. Angiogenesisbegins with the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

[0459] As persistent, unregulated angiogenesis occurs during tumordevelopment and metastasis, the treatment methods of this invention maybe used in combination with any one or more “anti-angiogenic” therapies.Exemplary anti-angiogenic agents that are useful in connection withcombined therapy are listed in Table C. Each of the agents listedtherein is exemplary and by no means limiting. TABLE C Inhibitors andNegative Regulators of Angiogenesis Substances References AngiostatinO'Reilly et al., 1994 Endostatin O'Reilly et al., 1997 l6kDa prolactinfragment Ferrara et al., 1991; Clapp et al., 1993; D'Angelo et al.,1995; Lee et al., 1998 Laminin peptides Kleinman et al., 1993; Yamamuraet al., 1993; Iwamoto et al., 1996; Tryggvason, 1993 Fibronectinpeptides Grant et al., 1998; Sheu et al., 1997 Tissue metalloproteinaseinhibitors Sang, 1998 (TIMP 1, 2, 3, 4) Plasminogen activator inhibitorsSoff et al., 1995 (PAI-1, -2) Tumor necrosis factor α (highFrater-Schroder et al., 1987 dose, invitro) TGF-β1 RayChadhury andD'Amore, 1991; Tada et al., 1994 Interferons (IFN-α, -β, γ) Moore etal., 1998; Lingen et al., 1998 ELR-CXC Chemokines: Moore et al., 1998;Hiscox and Jiang, 1997; Coughlin IL-12; SDF-1; MIG; Platelet factor 4 etal., 1998; Tanaka et al., 1997 (PF-4); IP-10 Thrombospondin (TSP) Goodet al., 1990; Frazier, 1991; Bornstein, 1992; Tolsma et al., 1993;Sheibani and Frazier, 1995; Volpert et al., 1998 SPARC Hasselaar andSage, 1992; Lane et al., 1992; Jendraschak and Sage, 19962-Methoxyoestradiol Fotsis et al., 1994 Proliferin-related proteinJackson et al., 1994 Suramin Gagliardi et al., 1992; Takano et al.,1994; Waltenberger et al., 1996; Gagliardi et al., 1998; Manetti et al.,1998 Thalidomide D'Amato et al., 1994; Kenyon et al., 1997 Wells, 1998Cortisone Thorpe et al., 1993 Folkman et al., 1983 Sakamoto et al., 1986Linomide Vukanovic et al., 1993; Ziche et al, 1998; Nagler et al., 1998Fumagillin (AGM-1470; TNP-470) Sipos et al., 1994; Yoshida et al., 1998Tamoxifen Gagliardi and Collins, 1993; Linder and Borden, 1997; Haran etal., 1994 Korean mistletoe extract Yoon et al., 1995 (Viscum albumcoloratum) Retinoids Oikawa et al., 1989; Lingen et al., 1996; Majewskiet al. 1996 CM101 Hellerqvist et al., 1993; Quinn et al., 1995; Wamil etal., 1997; DeVore et al., 1997 Dexamethasone Hori et al., 1996; Wolff etal., 1997 Leukemia inhibitory factor (LIF) Pepper et al., 1995

[0460] A certain preferred component for use in inhibiting angiogencsisis a protein named “angiostatin”. This component is disclosed in U.S.Pat. Nos. 5,776,704; 5,639,725 and 5,733,876, each incorporated hereinby reference. Angiostatin is a protein having a molecular weight ofbetween about 38 kD and about 45 kD, as determined by reducingpolyacrylamide gel electrophoresis, which contains approximately Kringleregions 1 through 4 of a plasminogen molecule. Angiostatin generally hasan amino acid sequence substantially similar to that of a fragment ofmurine plasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule.

[0461] The amino acid sequence of angiostatin varies slightly betweenspecies. For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

[0462] Certain anti-angiogenic therapies have already been shown tocause tumor regressions, and angiostatin is one such agent. Endostatin,a 20 kDa COOH-terminal fragment of collagen XVIII, the bacterialpolysaccharide CM101, and the antibody LM609 also have angiostaticactivity. However, in light of their other properties, they are referredto as anti-vascular therapies or tumor vessel toxins, as they not onlyinhibit angiogenesis but also initiate the destruction of tumor vesselsthrough mostly undefined mechanisms. Their combination with the presentinvention is clearly envisioned.

[0463] Angiostatin and endostatin have become the focus of intensestudy, as they are the first angiogenesis inhibitors that havedemonstrated the ability to not only inhibit tumor growth but also causetumor regressions in mice. There are multiple proteases that have beenshown to produce angiostatin from plasminogen including elastase,macrophage met alloelastase (MME), matrilysin (MMP-7), and 92 kDagelatinase B/type IV collagenase (MMP-9).

[0464] MME can produce angiostatin from plasminogen in tumors andgranulocyte-macrophage colony-stimulating factor (GMCSF) upregulates theexpression of MME by macrophages inducing thc production of angiostatin.The role of MME in angiostatin generation is supported by the findingthat MME is in fact expressed in clinical samples of hepatocellularcarcinomas from patients. Another protease thought to be capable ofproducing angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown toproduce angiostatin-like fragments from plasminogen in vitro.

[0465] The mechanism of action for angiostatin is currently unclear, itis hypothesized that it binds to an unidentified cell surface receptoron endothelial cells inducing endothelial cell to undergo programmedcell death or mitotic arrest. Endostatin appears to be an even morepowerful anti-angiogenesis and anti-tumor agent although its biology ismuch less clear. Endostatin is effective at causing regressions in anumber of tumor models in mice. Tumors do not develop resistance toendostatin and, after multiple cycles of treatment, tumors enter adormant state during which they do not increase in volume. In thisdormant state, the percentage of tumor cells undergoing apoptosis wasincreased, yielding a population that essentially stays the same size.Endostatin is also thought to bind an unidentified endothelial cellsurface receptor that mediates its effect.

[0466] CM101 is a bacterial polysaccharide that has been wellcharacterized in its ability to induce neovascular inflammation intumors. CM101 binds to and cross-links receptors expressed ondedifferentiated endothelium that stimulates the activation of thecomplement system. It also initiates a cytokine-driven inflammatoryresponse that selectively targets the tumor. It is a uniquelyantipathoangiogenic agent that downregulates the expression VEGF and itsreceptors. CM101 is currently in clinical trials as an anti-cancer drug,and can be used in combination herewith.

[0467] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also beused in combination with the present invention. These are bothangiogenesis inhibitors that associate with heparin and are found inplatelet al.-granules. TSP-1 is a large 450 kDa multi-domainglycoprotein that is constituent of the extracellular matrix. TSP-1binds to many of the proteoglycan molecules found in the extracellularmatrix including, HSPGs, fibronectin, laminin, and difl′ercnt types ofcollagen. TSP-1 inhibits endothelial cell migration and proliferation invitro and angiogenesis in vivo. TSP-1 can also suppress the malignantphenotype and tumorigenesis of transformed endothelial cells. The tumorsuppressor gene p53 has been shown to directly regulate the expressionof TSP-1 such that, loss of p53 activity causes a dramatic reduction inTSP-1 production and a concomitant increase in tumor initiatedangiogenesis.

[0468] PF4 is a 70aa protein that is member of the CXC ELR- family ofchemokines that is able Ito potently inhibit endothelial cellproliferation in vitro and angiogenesis in vivo. PF4 administeredintratumorally or delivered by an adenoviral vector is able to cause aninhibition of tumor growth.

[0469] Interferons and met alloproteinase inhibitors are two otherclasses of naturally occurring angiogenic inhibitors that can becombined with the present invention. The anti-endothelial activity ofthe interferons has been known since the early 1980s, however, themechanism of inhibition is still unclear. It is known that they caninhibit endothelial cell migration and that they do have someanti-angiogenic activity in vivo that is possibly mediated by an abilityto inhibit the production of angiogenic promoters by tumor cells.Vascular tumors in particular are sensitive to interferon, for example,proliferating hemangiomas can be successfully treated with IFNα.

[0470] Tissue inhibitors of met alloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix met alloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in combined treatmentprotocols with the present invention. MMPs play a key role in theangiogenic process as they degrade the matrix through which endothelialcells and fibroblasts migrate when extending or remodeling the vascularnetwork. In fact, one member of the MMPs, MMP-2, has been shown toassociate with activated endothelium through the integrin αvβ3presumably for this purpose. If this interaction is disrupted by afragment of MMP-2, then angiogenesis is downregulated and in tumorsgrowth is inhibited.

[0471] There are a number of pharmacological agents that inhibitangiogenesis, any one or more of which may be used in combination withthe present invention. These include AGM-1470/TNP-470, thalidomide, andcarboxyamidotriazole (CAI). Fumagillin was found to be a potentinhibitor of angiogenesis in 1990, and since then the syntheticanalogues of fumagillin, AGM-1470 and TNP-470 have been developed. Bothof these drugs inhibit endothelial cell proliferation in vitro andangiogenesis in vivo. TNP-470 has been studied extensively in humanclinical trials with data suggesting that long-term administration isoptimal.

[0472] Thalidomide was originally used as a sedative but was found to bea potent teratogen and was discontinued. In 1994 it was found thatthalidomide is an angiogenesis inhibitor. Thalidomide is currently inclinical trials as an anti-cancer agent as well as a treatment ofvascular eye diseases.

[0473] CAI is a small molecular weight synthetic inhibitor ofangiogenesis that acts as a calcium channel blocker that prevents actinreorganization, endothelial cell migration and spreading on collagen IV.CAI inhibits neovascularization at physiological attainableconcentrations and is well tolerated orally by cancer patients. Clinicaltrials with CAI have yielded disease stabilization in 49% of cancerpatients having progressive disease before treatment.

[0474] Cortisone in the presence of heparin or heparin fragments wasshown to inhibit tumor growth in mice by blocking endothelial cellproliferation. The mechanism involved in the additive inhibitory effectof the steroid and heparin is unclear although it is thought that theheparin may increase the uptake of the steroid by endothelial cells. Themixture has been shown to increase the dissolution of the basementmembrane underneath newly formed capillaries and this is also a possibleexplanation for the additive angiostatic effect. Heparin-cortisolconjugates also have potent angiostatic and anti-tumor effects activityin vivo.

[0475] Further specific angiogenesis inhibitors, including, but notlimited to, Anti-Invasive Factor, retinoic acids and paclitaxel (U.S.Pat. No. 5,716,981; incorporated herein by reference); AGM-1470 (Ingberet al., 1990; incorporated herein by reference); shark cartilage extract(U.S. Pat. No. 5,618,925; incorporated herein by reference); anionicpolyamide or polyurea oligomers (U.S. Pat. No. 5,593,664; incorporatedherein by reference); oxindole derivatives (U.S. Pat. No. 5,576,330;incorporated herein by reference); estradiol derivatives (U.S. Pat. No.5,504,074; incorporated herein by reference); and thiazolopyrimidinederivatives (U.S. Pat. No. 5,599,813; incorporated herein by reference)are also contemplated for use as anti-angiogenic compositions for thecombined uses of the present invention.

[0476] Compositions comprising an antagonist of an α_(v)β₃ integrin mayalso be used to inhibit angiogenesis in combination with the presentinvention. As disclosed in U.S. Pat. No. 5,766,591 (incorporated hereinby reference), RGD-containing polypeptides and salts thereof, includingcyclic polypeptides, are suitable examples of cc,p, integrinantagonists.

[0477] The antibody LM609 against the α_(v)β₃ integrin also inducestumor regressions. Integrin α_(v)β₃ antagonists, such as LM609, induceapoptosis of angiogenic endothelial cells leaving the quiescent bloodvessels unaffected. LM609 or other α_(v)β₃ antagonists may also work byinhibiting the interaction of α_(v)β₃ and MMP-2, a proteolytic enzymethought to play an important role in migration of endothelial cells andfibroblasts.

[0478] Apoptosis of the angiogenic endothelium in this case may have acascade effect on the rest of the vascular network. Inhibiting the tumorvascular network from completely responding to the tumor's signal toexpand may, in fact, initiate the partial or fuill collapse of thenetwork resulting in tumor cell death and loss of tumor volume. It ispossible that endostatin and angiostatin function in a similar fashion.The fact that LM609 does not affect quiescent vessels but is able tocause tumor regressions suggests strongly that not all blood vessels ina tuinor need to be targeted for treatment in order to obtain ananti-tumor effect.

[0479] Targeted or non-targeted angiopoietins, preferablyangiopoietin-2, may also be used in combination with the presentinvention (see discussion below for targeted angiopoietins).Angiopoietin-2 (SEQ ID NO:3 and SEQ ID NO:4) is a ligand for Tie2 andgenerally counteracts blood vessel maturation/stability mediated byangiopoletin-1. It is thus an antagonist of angiopoictin-1, and acts todisturb capillary structure. In the absence of another angiogenic signal(such as VEGF), angiopoietin-2 causes vessels to destabilize and becomeimmature (Ilolash el al., 1999; incorporated herein by reference).Provision of targeted or non-targcted angiopoietin-2 in connection withthe methods of the present invention is thus contemplated, particularlyin tumors with low VEGF levels and/or in combination with VEGFinhibition. Manipulation of angiopoietin-1 and two new angiopoictins,angiopoietin-3 (mouse) and angiopoietin-4 (human), could also be used inconjunction with this invention.

[0480] Other methods of therapeutic intervention based upon alteringsignaling through the Tie2 receptor can also be used in combination withthe present invention, such as using a soluble Tie2 receptor capable ofblocking Tie2 activation (Lin et al., 1998). Delivery of such aconstruct using recombinant adenoviral gene therapy has been shown to beeffective in treating cancer and reducing metastases (Lin et al., 1998).

[0481] J3. Apoptosis-Inducing Agents

[0482] Therapeutic agent-targeting agent treatment may also be combinedwith treatment methods that induce apoptosis in any cells within thetumor, including tumor cells and tumor vascular endothelial cells.Although many anti-cancer agents may have, as part of their mechanism ofaction, an apoptosis-inducing effect, certain agents have beendiscovered, designed or selected with this as a primary mechanism, asdescribed below.

[0483] A number of oncogenes have been described that inhibit apoptosis,or programmed cell death. Exemplary oncogenes in this category include,but are not limited to, bcr-abl, bcl-2 (distinct from bcl-1, cyclin D1;GenBank accession numbers M14745, X06487; U.S. Pat. No. 5,650,491; and5,539,094; each incorporated herein by reference) and family membersincluding Bcl-x1, Mcl-1, Bak, A1, A20. Overexpression of bcl-2 was firstdiscovered in T cell lymphomas. bcl-2 functions as an oncogene bybinding and inactivating Bax, a protein in the apoptotic pathway.Inhibition of bcl-2 function prevents inactivation of Bax, and allowsthe apoptotic pathway to proceed. Thus, inhibition of this class ofoncogenes, e.g., using antisense nucleotide sequences, is contemplatedfor use in the present invention in aspects wherein enhancement ofapoptosis is desired (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

[0484] Many forms of cancer have reports of mutations in tumorsuppressor genes, such as p53.

[0485] Inactivation of p53 results in a failure to promote apoptosis.With this failure, cancer cells progress in tumorigenesis, rather thanbecome destined for cell death. Thus, provision of tumor suppressors isalso contemplated for use in the present invention to stimulate celldeath. Exemplary tumor suppressors include, but are not limited to, p53,Retinoblastoma gene (Rb), Wilm's tumor (WT1), bax alpha,interleukin-lb-converting enzyme and family, MEN-1 gene,neurofibromatosis, type I (NF1), cdk inhibitor p16, colorectal cancergene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumorsuppressor gene (MTS-1), BRCA1 and BRCA2.

[0486] Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469;5,677,178; and 5,756,455; each incorporated herein by reference),Retinoblastoma, BRCA1 (U.S. Pat. Nos. 5,750,400; 5,654,155; 5,710,001;5,756,294; 5,709,999; 5,693,473; 5,753,441; 5,622,829; and 5,747,282;each incorporated herein by reference), MEN-1 (GenBank accession numberU93236) and adenovirus E1A (U.S. Pat. No. 5,776,743; incorporated hereinby reference) genes.

[0487] Other compositions that may be used include genes encoding thetumor necrosis factor related apoptosis inducing ligand termed TRAIL,and the TRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated hereinby reference); the 24 kD apoptosis-associated protease of U.S. Pat. No.5,605,826 (incorporated herein by reference); Fas-associated factor 1,FAF1 (U.S. Pat. No. 5,750,653; incorporated herein by reference). Alsocontemplated for use in these aspects of the present invention is theprovision of interleukin-1β-converting enzyme and family members, whichare also reported to stimulate apoptosis.

[0488] Compounds such as carbostyril derivatives (U.S. Pat. Nos.5,672,603; and 5,464,833; each incorporated herein by reference);branched apogenic peptides (U.S. Pat. No. 5,591,717; incorporated hereinby reference); phosplhotyrosine inhibitors and non-hydrolyzablephosphotyrosine analogs (U.S. Pat. Nos. 5,565,491; and 5,693,627; eachincorporated herein by reference); agonists of RXR retinoid receptors(U.S. Pat. No. 5,399,586; incorporated herein by reference); and evenantioxidants (U.S. Pat. No. 5,571,523; incorporated herein by reference)may also be used. Tyrosine kinase inhibitors, such as genistein, mayalso be linked to ligands that target a cell surface receptor (U.S. Pat.No. 5,587,459; incorporated herein by reference).

[0489] J4. Immunotoxins and Coaguligands

[0490] The naked anti-aminophospholipid antibody treatment methods ofthe invention may be used in combination with immunotoxins (ITs) and/orcoaguligands in which the targeting portion thereof, e.g., antibody orligand, is directed to a relatively specific marker of the tumor cells,tumor vasculature or tumor stroma. In common with the chemotherapeuticand anti-angiogenic agents discussed above, the use of ananti-aminophospholipid antibody in combination with a targeted toxin orcoagulant will generally result in the distinct agents being directedagainst different targets within the tumor environment. This shouldresult in additive, markedly greater than additive or even synergisticanti-tumor results.

[0491] Generally speaking, antibodies or ligands for use in theseadditional aspects of the invention will preferably recognize accessibletumor antigens that are preferentially, or specifically, expressed inthe tumor site. The antibodies or ligands will also preferably exhibitproperties of high affinity; and the antibodies, ligands or conjugatesthereof, will not exert significant in vivo side effects againstlife-sustaining normal tissues, such as one or more tissues selectedfrom heart, kidney, brain, liver, bone marrow, colon, breast, prostate,thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas,skin, or other life-sustaining organ or tissue in the human body. Theterm “significant side effects”, as used herein, refers to an antibody,ligand or antibody conjugate, that, when administered in vivo, willproduce only negligible or clinically manageable side effects, such asthose normally encountered during chemotherapy.

[0492] At least one binding region of these second anti-cancer agentsemployed in combination with the anti-aminophospholipid antibodies ofthe invention will be a component that is capable of delivering a toxinor coagulation factor to the tumor region, i.e., capable of localizingwithin a tumor site. Such targeting agents may be directed against acomponent of a tumor cell, tumor vasculature or tumor stroma. Thetargeting agents will generally bind to a surface-expressed,surface-accessible or surface-localized component of a tumor cell, tumorvasculature or tumor stroma. However, once tumor vasculature and tumorcell destruction begins, internal components will be released, allowingadditional targeting of virtually any tumor component.

[0493] Many tumor cell antigens have been described, any one which couldbe employed as a target in connection with the combined aspects of thepresent invention. Appropriate tumor cell antigens for additionalimmunotoxin and coaguligand targeting include those recognized by theantibodies B3 (U.S. Pat. No. 5,242,813; incorporated herein byreference; ATCC HB 10573); KSI/4 (U.S. Pat. No. 4,975,369; incorporatedherein by reference; obtained from a cell comprising the vectors NRRLB-18356 and/or NRRL B-18357); 260F9 (ATCC HB 8488); and D612 (U.S. Pat.No. 5,183,756; incorporated herein by reference; ATCC HB 9796). One mayalso consult the ATCC Catalogue of any subsequent year to identify otherappropriate cell lines producing anti-tumor cell antibodies.

[0494] For tumor vasculature targeting, the targeting antibody or ligandwill often bind to a marker expressed by, adsorbed to, induced on orotherwise localized to the intratumoral blood vessels of a vascularizedtumor. Appropriate expressed target molecules include, for example,endoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA (Liu et al.,1997), a TIE, a ligand reactive with LAM-1, a VEGF/VPF receptor, an FGFreceptor, α_(v)α₃ integrin, pleiotropin and endosialin. Suitableadsorbed targets are those such as VEGF, FGF, TGFβ, HGF, PF4, PDGF,TIMP, a ligand that binds to a TIE and tumor-associated fibronectinisoforms. Antigens naturally and artificially inducible by cytokines andcoagulants may also be targeted, such as ELAM-1, VCAM-1, ICAM-1, aligand reactive with LAM-1, endoglin, and even MHC Class II(cytokine-inducible, e.g., by IL-1, TNF-α, IFN-γ, IL-4 and/or TNF-β);and E-selectin, P-selectin, PDGF and ICAM-1 (coagulant-inducible e.g.,by thrombin, Factor IX/IXa, Factor X/Xa and/or plasmin).

[0495] The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use ofimmunotoxins directed against expressed, adsorbed, induced or localizedmarkers of tumor vasculature: U.S. Pat. Nos. 5,855,866; 5,776,427;5,863,538; 5,660,827; 5,855,866 and 5,877,289; and U.S. application Ser.Nos. 07/846,349; 08/295,868 (U.S. Pat. No. 5,___,___; Issue Fee paid);08/350,212 (U.S. Pat. No. 5,___,___; Issue Fee paid); 08/457,869 (Noticeof Allowance Received); 08/482,369 (U.S. Pat. No. 5,___,___; Issue Feepaid); 08/487,427 (U.S. Pat. No. 5,___,___; Issue Fee paid); and08/479,727 (U.S. Pat. No. 5,___,___; Issue Fee paid).

[0496] Suitable tumor stromal targets include components of the tumorextracellular matrix or stroma, or components those bound therein;including basement membrane markers, type IV collagen, laminin, heparansulfate, proteoglycan, fibronectins, activated platelets, LIBS andtenascin. A preferred target for such uses is RIBS.

[0497] The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use oftumor stromal targeting agents: U.S. Pat. No. 5,877,289 and U.S.application Ser. Nos. 08/482,369 (U.S. Pat. No. 5,___,___; Issue Feepaid); 08/487,427 (U.S. Pat. No. 5,___,___; Issue Fee paid); and08/479,727 (U.S. Pat. No. 5,___,___; Issue Fee paid).

[0498] In certain applications, the second anti-cancer therapeutics willbe antibodies or ligands operatively attached to cytotoxic or otherwiseanti-cellular agents having the ability to kill or suppress the growthor cell division of endothelial cells. Suitable anti-cellular agentsinclude chemotherapeutic agents and radioisotopes, as well ascytotoxins. Exemplary chemotherapeutic agents include: steroids;cytokines; anti-metabolites, such as cytosine arabinoside, fluorouracil,methotrexate or aminopterin; anthracyclines; mitomycin C; vincaalkaloids; antibiotics; demecolcine; etoposide, mithramycin; andanti-tumor alkylating agents, such as chlorambucil or melphalan.

[0499] In most combined therapeutic applications, toxin moieties will beprefcrrcd, due to the much greater ability of most toxins to deliver acell killing effect, as compared to other potential agents. Therefore,preferred anti-cellular agents for second therapeutics are plant-,fungus- or bacteria-derived toxins. Exemplary toxins includeepipodophyllotoxins; bacterial endotoxin or the lipid A moiety ofbacterial endotoxin; ribosome inactivating proteins, such as saporin orgelonin; et-sarcin; aspergillin; restrictocin; ribonucleases, such asplacental ribonuclease; diphtheria toxin and pseudomonas exotoxin.

[0500] Certain preferred toxins are gelonin and the A chain toxins, suchas ricin A chain. The most preferred toxin moiety is often ricin A chainthat has been treated to modify or remove carbohydrate residues, socalled “deglycosylated A chain” (dgA). Deglycosylated ricin A chain ispreferred because of its extreme potency, longer half-life, and becauseit is economically feasible to manufacture it a clinical grade andscale. Recombinant and/or truncated ricin A chain may also be used.

[0501] Other agents for use with immunoconjugates for targeting tumorvasculature or tumor stroma are the angiopoietins. The angiopoietins,like the members of the VEGF family, are growth factors largely specificfor vascular endothelium (Davis and Yancopoulos, 1999; Holash et al.,1999; incorporated herein by reference). The angiopoietins firstdescribed were a naturally occurring agonist, angiopoietin-1 (Ang-1; SEQID NO:1 and SEQ ID NO:2), and a naturally occurring antagonist,angiopoietin-2 (Ang-2; SEQ ID NO:3 and SEQ ID NO:4), both of which actby means of the endothelial cell tyrosine kinase receptor, Tie2.

[0502] Two new angiopoietins, angiopoietin-3 (mouse) and angiopoietin-4(human) have also been identified (Valenzuela et al., 1999).Angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4appears to function as an agonist (Valenzuela et al., 1999). A proteintermed angiopoietin-3 was also cloned from human heart and reported notto have mitogenic effects on endothelial cells (Kim el al., 1999).Fusion proteins of angiopoietin-1 and angiopoictin-2 have also beencreated, as exemplified by the stable Ang-1-Alig-2 fusion proteinincluded herein as SEQ ID NO:5.

[0503] Whereas VEGF is necessary for the early stages of vasculardevelopment, angiopoietin-1 is generally required for the later stagesof vascular remodeling. Angiopoietin-1 is thus a maturation orstabilization factor, which converts immature vessels to mature vessels.

[0504] Angiopoietin-1 has been shown to augment revascularization inischemic tissue (Shyu et al., 1998) and to increase the survival ofvascular networks exposed to either VEGF or a form of aFGF(Papapetropoulos et al., 1999). These authors also showed thatangiopoietin-1 prevents apoptotic death in HUVEC triggered by withdrawalof the same form of aFGF (Papapetropoulos et al., 1999). Such data areconsistent with the direct role of angiopoietin-1 on human endothelialcells and its interaction with other angiogenic molecules to stabilizevascular structures by promoting the survival of differentiatedendothelial cells.

[0505] Angiopoietin-2 is a preferred agent for use in targetedcombination therapy, particularly in tumors with low VEGF levels and/orin combination with VEGF inhibition. Angiopoietin-2 is also a ligand forTie2, but generally counteracts blood vessel maturation/stabilitymediated by angiopoietin-1. It is thus an antagonist of angiopoietin-1,and acts to disturb capillary structure. However, as angiopoietin-2renders endothelial cells responsive to angiogenic stimuli, it caninitiate neovascularization in combination with other appropriatesignals, particularly VEGF (Asahara et al., 1998; Holash et al., 1999;incorporated herein by reference).

[0506] In the absence of another angiogenic signal, angiopoietin-2causes vessels to destabilize and become immature. In the presence of astimulus, such as VEGF, angiopoietin-2 promotes angiogenesis. Indeed,the angiogenic effects of a number of regulators are believed to beachieved, at least in part, through the regulation of an autocrine loopof angiopoietin-2 activity in microvascular endothelial cells (Mandriotaand Pepper, 1998).

[0507] Angiopoietin-2 expression in tumor tissue has been reported(Tanaka et biL., 1999), where it presumably acts in combination withVEGF to promote angiogenesis (Stratmann el al., 1998; Holash et al.,1999). However, as angiopoietin-2 provides a negative signal when VEGFis low or absent, provision of angiopoietin-2 can be a usefultherapeutic approach. Angiopoietin-2 can be administered as a proteintherapeutic or via gene therapy (see above), or in a tumor-targetedform. Although all types of targeted angiopoietin-2 constructs areenvisioned for use in the combined therapy aspects of the invention,currently preferred agents for targeting angiopoietin-2 to the tumor arethose that bind to aminophospholipids, including anti-PS antibodies andannexins.

[0508] For tumor targeting and treatment with immunotoxins, thefollowing patents and patent applications are specifically incorporatedherein by reference for the purposes of even further supplementing thepresent teachings regarding anticellular and cytotoxic agents: U.S. Pat.Nos. 5,855,866; 5,776,427; 5,863,538; and 5,660,827; and U.S.application Ser. Nos.07/846,349; 08/295,868 (U.S. Pat. No. 5,___,___;Issue Fee paid); 08/350,212 (U.S. Pat. No. 5,___,___; Issue Fee paid);and 08/457,869 (Notice of Allowance Received).

[0509] The second, targeted agent for optional use with theanti-aminophospholipid antibodies of the invention may also comprise atargeted component that is capable of promoting coagulation, i.e., a“coaguligand”. Here, the targeting antibody or ligand may be directly orindirectly, e.g., via another antibody, linked to a factor that directlyor indirectly stimulates coagulation.

[0510] Preferred coagulation factors for such uses are Tissue Factor(TF) and TF derivatives, such as truncated TF (tTF), dimeric andmultimeric TF, and mutant TF deficient in the ability to activate FactorVII. Other suitable coagulation factors include vitamin K-dependentcoagulants, such as Factor II/Ila, Factor VII/VNla, Factor IX/IXa andFactor X/Xa; vitamin K-dependent coagulation factors that lack the Glamodification; Russell's viper venom Factor X activator;platelet-activating compounds, such as thromboxane A₂ and thromboxane A₂synthase; and inhibitors of fibrinolysis, such as ot2-antiplasmin.

[0511] Tumor targeting and treatment with coaguligands is described inthe following patents and patent applications, each of which arespecifically incorporated herein by reference for the purposes of evenfurther supplementing the present teachings regarding coaguligands andcoagulation factors: U.S. Pat. Nos. 5,855,866 and 5,877,289; U.S.application Ser. Nos. 07/846,349; 08/350,212 (U.S. Pat. No. 5,___,___;Issue Fee paid); 08/482,369 (U.S. Pat. 5,___,___; Issue Fee paid);08/487,427 (U.S. Pat. No. 5,___,___; Issue Fee paid); and 08/479,727(U.S. Pat. No. 5,___,___; Issue Fee paid).

[0512] As somewhat wider distribution of a coagulating agent will not beassociated with severe side effects, there is a less stringentrequirement imposed on the targeting element of coaguligands than withimmunotoxins. Therefore, to achieve specific targeting means thatcoagulation is promoted in the tumor vasculature relative to thevasculature in non-tumor sites. Thus, specific targeting of acoaguligand is a functional term, rather than a purely physical termrelating to the biodistribution properties of the targeting agent. It isnot unlikely that useful targets may be not be entirelytumor-restricted, and that targeting ligands that are effective topromote tumor-specific coagulation may nevertheless be safely found atother sites of the body following administration, as occurs with VCAM-1.

[0513] The preparation of immunotoxins is generally well known in theart (see, e.g., U.S. Pat. No. 4,340,535, incorporated herein byreference). Each of the following patents and patent applications arefurther incorporated herein by reference for the purposes of evenfurther supplementing the present teachings regarding immunotoxingeneration, purification and use: U.S. Pat. Nos. 5,855,866; 5,776,427;5,863,538; and 5,660,827; and U.S. application Ser. Nos. 07/846,349;08/295,868 (U.S. Pat. No. 5,___,___; Issue Fee paid); 08/350,212 (U.S.Pat. No. 5,___,___; Issue Fee paid); and 08/457,869 (Notice of AllowanceReceived).

[0514] In the preparation of immunotoxins, advantages may be achievedthrough the use of certain linkers. For example, linkers that contain adisulfide bond that is sterically “hindered” are often preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action. It is generally desiredto have a conjugate that will remain intact under conditions foundeverywhere in the body except the intended site of action, at whichpoint it is desirable that the conjugate have good “release”characteristics.

[0515] Depending on the specific toxin compound used, it may benecessary to provide a peptide spacer operatively attaching thetargeting agent and the toxin compound, wherein the peptide spacer iscapable of folding into a disulfide-bonded loop structure. Proteolyticcleavage within the loop would then yield a heterodimeric polypeptidewherein the targeting agent and the toxin compound are linked by only asingle disulfide bond.

[0516] When certain other toxin compounds are utilized, a non-cleavablepeptide spacer may be provided to operatively attach the targeting agentand the toxin compound. Toxins that may be used in conjunction withnon-cleavable peptide spacers are those that may, themselves, beconverted by proteolytic cleavage, into a cytotoxic disulfide-bondedform. An example of such a toxin compound is a Pseudonomas exotoxincompound.

[0517] A variety of chemotherapeutic and other pharmacological agentscan also be successfully conjugated to antibodies or targeting ligands.Exemplary antineoplastic agents that have been conjugated to antibodiesinclude doxorubicin, daunomycin, methotrexate and vinblastine. Moreover,the attachment of other agents such as neocarzinostatin, macromycin,trenimon and α-amanitin has been described (see U.S. Pat. No. 5,855,866;and U.S. Pat. No. 5,___,___ (application Ser. No. 08/350,212, Issue Feepaid) and references incorporated therein.

[0518] In light of one of the present inventors earlier work, thepreparation of coaguligands is now also easily practiced. The operableassociation of one or more coagulation factors with a targeting agentmay be a direct linkage, such as those described above for theimmunotoxins. Alternatively, the operative association may be anindirect attachment, such as where the targeting agent is operativelyattached to a second binding region, preferably and antibody or antigenbinding region of an antibody, that binds to the coagulation factor. Thecoagulation factor should be attached to the targeting agent at a sitedistinct from its functional coagulating site, particularly where acovalent linkage is used to join the molecules.

[0519] Indirectly linked coaguligands are most often based uponbispecific antibodies. The preparation of bispecific antibodies is alsowell known in the art. One preparative method involves the separatepreparation of antibodies having specificity for the targeted tumorcomponent, on the one hand, and the coagulating agent on the other.Peptic F(ab′γ)₂ fragments from the two chosen antibodies are thengenerated, followed by reduction of each to provide separate Fab′ysFIfragments. The SH groups on one of the two partners to be coupled arethen alkylated with a cross-linking reagent, such aso-phenylenedimaleimide, to provide free maleimide groups on one partner.This partner may then be conjugated to the other by means of a thioetherlinkage, to give the desired F(ab′γ)₂ heteroconjugate (Glennie et al.,1987; incorporated herein by reference). Other approaches, such ascross-linking with SPDP or protein A may also be carried out.

[0520] Another method for producing bispecific antibodies is by thefusion of two hybridomas to form a quadroma. As used herein, the term“quadroma” is used to describe the productive fusion of two B cellhybridomas. Using now standard techniques, two antibody producinghybridomas are fused to give daughter cells, and those cells that havemaintained the expression of both sets of clonotype immunoglobulin genesare then selected.

[0521] A preferred method of generating a quadroma involves theselection of an enzyme deficient mutant of at least one of the parentalhybridomas. This first mutant hybridoma cell line is then fuised tocells of a second hybridoma that had been lethally exposed, e.g., toiodoacetamide, precluding its continued survival. Cell fusion allows forthe rescue of the first hybridoma by acquiring the gene for its enzymedeficiency from the lethally treated hybridoma, and the rescue of thesecond hybridoma through fusion to the first hybridoma. Preferred, butnot required, is the fusion of immunoglobulins of the same isotype, butof a different subclass. A mixed subclass antibody permits the use if analternative assay for the isolation of a preferred quadroma.

[0522] Microtiter identification embodiments, FACS, immunofluorescencestaining, idiotype specific antibodies, antigen binding competitionassays, and other methods common in the art of antibody characterizationmay be used to identify preferred quadromas. Following the isolation ofthe quadroma, the bispecific antibodies are purified away from othercell products. This may be accomplished by a variety of antibodyisolation procedures, known to those skilled in the art ofimmunoglobulin purification (see, e.g., Antibodies: A Laboratory Manual,1988; incorporated herein by reference). Protein A or protein Gsepharose colums are preferred.

[0523] In the preparation of both immunotoxins and coaguligands,recombinant expression may be employed. The nucleic acid sequencesencoding the chosen targeting agent, and toxin or coagulant, areattached in-frame in an expression vector. Recombinant expression thusresults in translation of the nucleic acid to yield the desiredtargeting agent-toxin/coagulant compound.

[0524] The following patents and patent applications are eachincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding coaguligand preparation,purification and use, including bispecific antibody coaguligands: U.S.Pat. Nos. 5,855,866 and 5,877,289; U.S. application Ser. Nos.07/846,349; 08/350,212 (U.S. Pat. No. No. 5,___,___; Issue Fee paid);08/273,567; 08/482,369; 08/485,482; and 08/472,631 (U.S. Pat. No.5,___,___; Issue Fee paid); 08/487,427 (U.S. Pat. No. 5,___,___; IssueFee paid); and 08/479,727 and 08/481,904 (U.S. Pat. No. 5,___,___; IssueFee paid).

[0525] Effective doses of immunotoxins and coaguligands for combined usewith the naked anti-aminophospholipid antibodies in the treatment ofcancer will be between about 0.1 mg/kg and about 2 mg/kg, andpreferably, of between about 0.8 mg/kg and about 1.2 mg/kg, whenadministered via the IV route at a frequency of about 1 time per week.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The physician responsible foradministration will determine the appropriate dose for the individualsubject.

[0526] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE I VCAM-1 Expression on Tumor and Normal Blood Vessels

[0527] A. Materials and Methods

[0528] 1. Materials

[0529] Na¹²⁵I was obtained from Amersham (Arlington Heights, Ill.).Dulbecco's modified Eagle's tissue culture medium (DMEM) and DulbeccoPBS containing Ca²⁺ and Mg²⁺ were obtained from Gibco (Grand Island,N.Y.). Fet al calf serum was obtained from Hyclone (Logan, Utah).O-phenylenediamine, hydrogen peroxide, 3-aminopropyltriethoxy-silane andsterile, endotoxin-free saline (0.9% NaCl in 100 ml of water) were fromSigma (St. Louis, Mo.). SMPT was from Pierce (Rockford, Ill.). Proplex Tcontaining factor VII (74 IU/ml), factor X and factor IX (17 IU/ml) waspurchased from Baxter Diagnostics Inc. (McGraw Park, Ill.). Chromogenicsubstrate, S-2765, for measuring factor Xa proteolytic activity wasobtained from Chromogenix (Franklin, Ohio). Purified factor Xa waspurchased from American Diagnostica (Greenwich, Conn.). 96 and 48 flatbottom microtiter plates were obtained from Falcon (Becton Dickinson andCo., Lincoln Park, N.J.). Sepharose-Protein G beads and S200 Superdexwere purchased from Pharmacia (Piscataway, N.J.). Recombinant murineIL-1α was purchased from R&D Systems (Minneapolis, Minn.).

[0530] 2. Antibodies

[0531] The MK2.7 hybridoma, secreting a rat IgGI antibody against murineVCAM-1, was obtained from the American Type Culture Collection (ATCC,Rockville, Md.; ATCC CRL 1909). The characterization of this anti-VCAM-1antibody has been reported by Miyake el al (1991, incorporated herein byreference). The R187 hybridoma, secreting a rat IgGI antibody againstmurine viral protein p30 gag, was also obtained from the ATCC, and wasused as an isotype matched control for the anti-VCAM-1 antibody.

[0532] Mouse monoclonal antibody, 10H110, against human tissue factorwas prepared as described in Morrissey et al. (1988), and in U.S.application Ser. No. 08/482,369, each incorporated herein by reference.

[0533] MECA 32, a pan anti-mouse vascular endothelial cell antibody, wasprepared as described by Leppink et al. (1989, incorporated herein byreference). MJ 7/18 rat IgG, reactive with murine endoglin, was preparedas described by Ge and Butcher (1994, incorporated herein by reference).The MECA 32 and MJ 7/18 antibodies served as positive controls forimmunohistochemical studies.

[0534] Rabbit anti-rat and rat anti-mouse secondary antibodiesconjugated with horseradish peroxidase (HRP) were purchased from Dako(Carpinteria, Calif.).

[0535] 3. Antibody Purification

[0536] Anti-VCAM-1 hybridoma, MK 2.7, and the irrelevant controlhybridoma, R187, were grown in bioreactors (Heraeus, Inc., Germany) for12 days. Supernatants were centrifuged, filtered through 0.22 μm filtersand loaded onto Sepharose-Protein G colums. IgG was eluted with citricacid buffer, pH 3.5, dialyzed into PBS and stored thereafter at 4° C. inthe same buffer. Purity was estimated by SDS-PAGE and was routinely>90%.Binding capacity of the purified anti-VCAM-1 antibody was assessedimmunohistochemically on frozen sections of L540 tumor and by cell-basedELISA using IL-1α stimulated bEnd.3 cells, as described herein below.

[0537] 4. Tumor-Bearing Mice and Immunohistochemistry

[0538] Male CB17 SClD mice (Charles River, Wilmington, Mass.) weighingapproximately 25 g were injected with 1×10⁷ L540 Hodgkin's lymphomacells subcutaneously into the right flank. Tumors were allowed to growto a size of 0.4-0.7 cm¹. Animals were anesthetized with metafane andtheir blood circulation was perfused with heparinized saline asdescribed by Burrows et al. (1992, incorporated herein by reference).The tumor and major organs were removed and snap-frozen in liquidnitrogen.

[0539] Cryostat sections of the tissues were cut, incubated with theanti-VCAM-1 antibody and stained immunohistochemically to detect VCAM-1.Rat IgG was detected using rabbit anti-rat IgG conjugated to HRPfollowed by development with carbazole (Fries et al., 1993).

[0540] B. Results

[0541] The blood vessels of the major organs and a tumor from micebearing subcutaneous L540 human Hodgkin's tumors were examinedimmunohistochemically for VCAM-1 expression using an anti-VCAM-1antibody. VCAM-1 expression on tumor blood vessels was more peripheralthan central. However, as demonstrated in Example VI and Example VII,the anti-VCAM -1 antibody and coaguligand were evidently binding toblood transporting vessels, as clearly shown by the ability of thecoaguligand to arrest blood flow in all tumor regions and to causedestruction of the intratumoral region.

[0542] Overall, VCAM-1 expression was observed on 20-30% of total tumorblood vessels stained by the anti-endoglin antibody, MJ 7/18. VCAM-1staining of the tumor vessels was largely observed on venules. VCAM-1expression was similar in tumors up to 1500 mm³, but larger tumorsappeared to have reduced staining, with 5-10% of MJ 7/18 positivevessels being positive for VCAM-1.

[0543] Constitutive vascular expression of VCAM-1 was found in heart andlungs in both tumor-bearing and normal animals (Table 1). In the heart,strong staining was observed on venules and veins. Approximately 10% ofMECA 32 positive vessels were positive for VCAM-1. Staining in lungendothelium was weak in comparison to heart and tumor, and was confinedto a few large blood vessels. Strong stromal staining was observed intestis where VCAM-1 expression was strictly extravascular. Similarfindings regarding constitutive VCAM-1 expression in rodent lung andtestis were previously reported (Fries et al., 1993). TABLE 1 Expressionof VCAM-1 on Endothelium in Tissues of L540 Tumor Bearing Mice andLocalization of Anti-VCAM-1 Antibody anti-VCAM-1 antibody Tissue VCAM-1expression^(a) localization^(b) Adrenal −^(c) − Brain Cerebellum − −Brain Cortex − − Heart ++ ++ Kidney − − Large Intestine − − Liver − −Lung + + Pancreas − − Small Intestine − − Spleen − − Testis −^(d) − L540Hodgkin's tumor +++ +++

EXAMPLE II Localization of Anti-VCAM-1 Antibody In Vivo

[0544] A. Methods

[0545] Male CB17 SClD mice (Charles River, Wilmington, Mass.) weighingapproximately 25 g were injected with 1×10⁷ L540 Hodgkin's lymphomacells subcutaneously into the right flank. Tumors were allowed to growto a size of 0.4-0.7 cm³.

[0546] Mice were injected intravenously with 30 μg/25 g body weight ofanti-VCAM-1 antibody, R187 antibody or corresponding coaguligands, in200 μl of saline. Two hours later, animals were anesthetized withmetafane and their blood circulation was perfused with heparinizedsaline as described (Burrows et al., 1992; incorporated herein byreference). The tumor and major organs were removed and snap-frozen inliquid nitrogen.

[0547] Cryostat sections of the tissues were cut and were stainedimmunohistochemically for the presence of rat IgG or TF. Rat IgG wasdetected using rabbit anti-rat IgG conjugated to HRP followed bydevelopment with carbazole (Fries et al., 1993). Coaguligand wasdetected using the 10H10 antibody that recognizes human tissue factor,followed by HRP-labeled anti-mouse IgG. 10H10 antibody does notcross-react detectably with murine tissue factor (Morrissey et al.,1988, incorporated herein by reference) or other murine proteins.

[0548] B. Results

[0549] Mice bearing subcutaneous L540 tumors were injected intravenouslywith anti-VCAM-1 antibody and, two hours later, the mice wereexsanguinated. The tumor and normal organs were removed and frozensections were prepared and examined immunohistochemically to determinethe location of the antibody. Serial sections of the tissues wereexamined. Localized rat IgG was detected by HRP-labeled anti-rat Ig; andmurine blood vessels were identified by pan-endothelial antibody, MECA32.

[0550] Anti-VCAM-1 antibody was detected on endothelium of tumor, heartand lung (Table 1). The intensity and number of stained vessels wasidentical to that on serial sections of the same tissues staineddirectly with anti-VCAM-1 antibody (Table 1). Staining was specific asno staining of endothelium was observed in the tumor and organs of miceinjected with a species isotype matched antibody of irrelevantspecificity, R187. No localization of anti-VCAM-1 antibody was found intestis or any normal organ except heart and lung.

EXAMPLE III Preparation of Anti-VCAM-1·tTF Coaguligand

[0551] An anti-VCAM-1·tTF conjugate or “coaguligand” was prepared asfollows. Truncated tissue factor (tTF), with an additional addedcysteine introduced at N-terminus (U.S. application Ser. No. 08/482,369,incorporated herein by reference), was expressed in E coli and purifiedas described by Stone et al. (1995, incorporated herein by reference).After purification, the sulfhydryl group of N′ cysteine-tTF wasprotected by reaction with Ellman's reagent. The tTF derivative wasstored in small volumes at −70° C.

[0552] To prepare the anti-VCAM-1 coaguligand, 5 ml of anti-VCAM-1antibody IgG (2 mg/ml) in PBS were mixed with 36 μl of SMPT (10 mM)dissolved in dry DMF and incubated at room temperature for 1 h. Themixture was filtered through a colum of Sephadex G25 equilibrated in PBScontaining 1 mM EDTA. The fractions containing the SMPT-derivatizedantibody were concentrated to 4 ml by ultrafiltration in an Amicon cellequipped with a 10,000 Da cut-off filter. Freshly thawed tTF derivativewas incubated with 30 μl of DTT (10 mM) in H₂O for 10 min. at roomtemperature and was filtered through a colum of Sephadex G25equilibrated in PBS containing 1 mM EDTA. The eluted fractionscontaining reduced tTF were concentrated by ultrafiltration undernitrogen to a final volume of 3 ml.

[0553] The reduced tTF was mixed with the SMPT-derivatized antibody andthe mixture was allowed to react for 24 h at room temperature. At theend of the incubation, the reaction mixture was resolved by gelfiltration on a colum of Superdex S200 equilibrated in PBS. Fractionscontaining anti-VCAM-1·tTF having a M_(r) of 180,000 and correspondingto one molecule of antibody linked to one molecule of tTF werecollected.

EXAMPLE IV Binding of Anti-VCAM-1 Coaguligand to Activated EndothelialCells

[0554] A. Methods

[0555] 1. lodination of 10H10 antibody

[0556] Anti-human tissue factor antibody, 10H10, was radiolabeled with¹²⁵I using Chloramine T as described by Bocci (1964, incorporated hereinby reference). The specific activity was approximately 10,000 cpm/pg, ascalculated from protein determinations measured by a Bradford assay(Bradford, 1976).

[0557] 2. Cells

[0558] L540 Hodgkin cells (L540 Cy), derived from a patient withend-stage disease, were prepared as described in Diehl et al. (1985,incorporated herein by reference), and were obtained from Prof. VolkerDiehl (Klinik fur Innere Medizin der Universitaet, Koeln, Germany).bEnd.3 cells (murine brain endothelioma) were prepared as described inBussolino et al. (1991) and Montesano et al (1990), each incorporatedherein by reference, and were obtained from Prof. Werner Risau (MaxPlanck Institute, Ba,d Nauheim, Germany).

[0559] 3. Tissue Culture

[0560] bEnd.3 cells and hybridomas were maintained in DMEM supplementedwith 10% fet al calf serum, 2 mM L-glutamine, 2 units/ml penicillin Gand 2 μg/ml streptomycin. L540 cells were maintained in RPMI 1640containing the same additives. All cells were subcultured once a week.bEnd.3 trypsinization was performed using 0.125% trypsin in PBS solutioncontaining 0.2% EDTA. For binding studies, cells were seeded at adensity of 5×104 cells/ml in 0.5 ml of medium in 48 well plates andincubated for 48-96 h. Medium was refreshed 24 h before each study.

[0561] 4. Binding of Coaguligand to Activated Endothelial Cells

[0562] Binding of the anti-VCAM-1 antibody and coaguligand to VCAM-1 onactivated bEnd.3 cells was determined using a cell based ELISA, asdescribed by Hahne (1993, incorporated herein by reference). bEnd.3cells were incubated with 10 units/ml of IL-la for 4 h at 37° C. in96-well microtiter plates. At the end of this incubation, medium wasreplaced by DPBS containing 2 mM Ca²⁺ and Mg²⁺ and 0.2% (w/v) gelatin asa carrier protein. The same buffer was used for dilution of antibodiesand for washing of cell monolayers between steps.

[0563] Cells were incubated with 4 μg/ml of anti-VCAM-1·tTF conjugate,anti-VCAM-1 antibody or control reagents for 2 h, and were then washedand incubated for 1 h with rabbit anti-rat IgG-HRP conjugate (1:500dilution). All steps were performed at room temperature. HRP activitywas measured by adding O-phenylenediamine (0.5 mg/ml) and hydrogenperoxide (0.03% w/v) in citrate-phosphate buffer, pH 5.5. After 30 min.,100 Vl of supernatant were transferred to 96 well plates, 100 μl of 0.18M H₂SO₄ were added and the absorbance was measured at 492 nm. Each studywas performed in duplicate and repeated at least twice.

[0564] 5. Detection of Coaguligand Bound to Endothelial cells

[0565] Anti-VCAM-1 coaguligand and appropriate controls were incubatedwith IL-1α stimulated bEnd.3 cells in 96-well microtiter plates, asdescribed above. Bound coaguligands were detected by identifying boththe tissue factor component and the rat IgG component bound to bEnd.3cells.

[0566] After removing the excess of unbound antibody, cells wereincubated with 100 μl/well of ¹²⁵I-labeled 10H10 antibody (0.2 μg/ml) or¹²⁵I-labeled rabbit anti-rat Ig (0.2 μg/ml) in binding buffer. After 2 hincubation at room temperature, cells were washed extensively anddissolved in 0.5 M of NaOH. The entire volume of 0.5 ml was transferredto plastic tubes and counted in a γ counter. Each study was performed induplicate and repeated at least twice.

[0567] B. Results

[0568] The ability of an anti-VCAM-1·tTF coaguligand to bind to IL-1αactivated murine bEnd.3 cells was determined by measuring the binding ofradioiodinated anti-TF antibody to coaguligand-treated cells in vitro.VCAM-1 expression by bEnd.3 cells is transiently inducible by IL-1α witha peak of VCAM-1 expression being obtained 4-6 h after addition of thecytokine (Hahne et al., 1993). Strong binding of the coaguligand toactivated bEnd.3 cells was observed (FIG. 1A).

[0569] At saturation, 8.7 fmoles of anti-TF antibody was bound to thecells, which is equivalent to 540,000 molecules of anti-TF antibody percell. Binding of the coaguligand was specific; no detectable bindingover background was observed with an isotype matched control coaguligandof irrelevant specificity. Binding of coaguligand to unstimulated cellswas about half of that to activated cells and is probably attributableto constitutive VCAM-1 expression by cultured endothelioma cells.

[0570] In further studies, the anti-VCAM-1·tTF coaguligand was found tobind as strongly as unconjugated anti-VCAM-1 antibody to activatedbEnd.3 cells, using detection by peroxidase-labeled anti rat IgG in theassay. This was done at both saturating and subsaturatingconcentrations. Thus, the conjugation procedure (Example III) did notdiminish antibody's capacity to bind to VCAM-1 on intact endothelialmonolayers.

EXAMPLE V Factor X Activation by Endothelial Cell-Bound Coaguligand

[0571] A. Methods

[0572] The activity of the anti-VCAM-1·tTF coaguligand bound toactivated bEnd.3 cells was determined indirectly by using a chromogenicassay to detect factor Xa (Schorer et al., 1985; Nawroth et al., 1985;each incorporated herein by reference). IL-1α-stimulated andunstimulated bEnd.3 cells were incubated with specific and controlcoaguligands in 96-well microtiter plates as described above. The cellswere washed with PBS containing 2 mg/ml of BSA and were incubated with150 μl/well of freshly prepared Proplex T solution diluted 1:20 in 50 mMTris-HCl (pH 8.1), 150 mM NaCl, 2 mg/ml BSA (tissue culture grade,endotoxin-free) and 2.5 mM CaCl₂. After incubation for 60 min. at 37°C., 100 μl were withdrawn from each well, transferred to 96-well platesand mixed with 100 μl of the same buffer containing 12.5 mM EDTA (pH8.1).

[0573] Chromogenic substrate S2765 for measuring factor Xa proteolyticactivity was added in 50 μl, giving a final concentration of 300 μM.′I′hc breakdown of the substrate was determined by reading theabsorbance at 405 nm over a 2 h period in a microplate reader (MolecularDevices, Palo Alto, Calif.).

[0574] Production of the chromogenic product was completely dependent onthe presence of Proplex T and bEnd.3 cells preincubated with thespecific coaguligand. Background hydrolysis of the substrate by ProplexT in the absence of cells was approximately 10% of the maximal value andwas subtracted from each determination. Free coaguligands diluted inProplex T solution were unable to generate factor Xa. The amount of Xagenerated was calculated by reference to a standard curve constructedwith known concentrations of purified factor Xa.

[0575] At the end of the study, cells were detached with trypsin-EDTAand counted. The results are expressed as the amount of factor Xagenerated per 10⁴ cells. Each study was performed in duplicate and wasrepeated at least 3 times.

[0576] B. Results

[0577] 1. Factor X Activation

[0578] Anti-VCAM-1·tTF coaguligand bound to IL-1α-activated bEnd.3 cellswas capable of specifically activating factor X. The rate of generationof factor Xa by anti-VCAM-1·tTF coated cells was 3.2 ng per 10⁴ cellsper hour, which is 7-10 fold higher than was observed with activatedcells treated with a control coaguligand of irrelevant specificity orwith tTF alone (FIG. IB). Anti-VCAM-1·tTF in the absence of cells hadundetectable factor X activating activity, confirming that cell bindingis essential for coaguligand activity.

[0579] Anti-VCAM-1·tTF bound to unstimulated bEnd.3 cells activatedfactor X at a rate of 1.6 ng per 10⁴ cells per hour. This rate is abouthalf that observed with the IL-lot-stimulated cells, in accordance withthe 50% lower amount of coaguligand that binds to unstimulated ascompared with stimulated cells. Similar results to those shown in FIG.1B were obtained in three separate studies.

[0580] 2. Effect of Endothelial Cell Permcabilization

[0581] Permeabilization of bEnd.3 monolayers with saponin after treatingthem with anti-VCAM -1·tTF coaguligand increased the ability of thebound coaguligand to activate factor X by about 30-fold (Table 2). Therate of factor Xa generation by unstimulated cells treated withanti-VCAM -1 tTF increased from 1.6 to 49.2 ng per 10⁴ cells per hourafter permeabilization, while that of IL-lu stimulated cells increasedfrom 3.2 to 98.8 ng per 10⁴ cells per hour. The factor Xa generatingactivity of the permeabilized cells was due to the bound coaguligandrather than to endogenous TF since permeabilized untreated cells orcells treated with control coaguligand of irrelevant specificity had lowfactor Xa generating activity (2 ng per 1 cells per hour).

[0582] These results indicate that the coaguligand is able to functionmore efficiently in the environment of a permeabilized cell. Possibly,permeabilization exposes negatively-charged phospholipids from withinthe cell that accelerate the formation of the coagulation-initiationcomplexes, or else prevents the inactivation of such complexes by TFPI.TABLE 2 Generation of Factor Xa by Anti-VCAM-1·tTF Bound to Intact orPermeabilized bEnd.3 cells (ng per 10⁴ cells per 60 min.) Intact cellsPermeabilized cells^(b) Treatment^(a) Control IL-1α Control IL-1α Buffer0.25^(c) 0.43 0.45 2.0 tTF 0.26 0.42 0.39 2.1 Control IgG·tTF 0.26 0.430.41 2.1 Anti-VCAM-1·tTF 1.64 3.17 49.2 98.8

EXAMPLE VI Tumor Blood Vessel Thrombosis by Anti-VCAM-1 Coaguligand

[0583] A . Methods

[0584] SCID mice bearing L540 tumors (0.4-0.7 cm³) were injectedintravenously with 40 μg (total protein) of anti-VCAM-1·tTF or R187·tTF.This dose corresponds to 32 μg of antibody and 8 μg of tTF. Otheranimals received equivalent quantities of free antibody, free tTF or amixture of both. Animals were anesthetized 4 or 24 h later and theirblood circulations were perfused with heparinized saline. The tumor andmajor organs were removed and were fixed in fodmalin andparaffin-embedded or snap-frozen for cryosectioning. Sections were cutthrough the center of the tissue or tumor. The number of thrombosed andnon-thrombosed blood vessels in 5 cross-sections were counted. Thepercentage of thrombosed vessels was calculated.

[0585] B. Results

[0586] 1. Thrombosis of Tumor Blood Vessels

[0587] This study shows that intravenous administration of theanti-VCAM-1-1·tTF coaguligand induces selective thrombosis of tumorblood vessels, as opposed to vessels in normal tissues, in tumor-bearingmice.

[0588] The anti-VCAM-1·tTF coaguligand was administered to mice bearingsubcutaneous L540 tumors of 0.4 to 0.6 cm in diameter. Beforecoaguligand injection, tumors were healthy, having a uniform morphologylacking regions of necrosis. The tumors were well vascularized and had acomplete absence of spontaneously thrombosed vessels or hemorrhages.Within four hours of coaguligand injection, 40-70% of blood vessels werethrombosed, despite the initial staining of only 20-30% of tumor bloodvessels shown in Example I. The thrombosed vessels contained occlusiveplatelet aggregates, packed erythrocytes and fibrin. In several regions,the blood vessels had ruptured, spilling erythrocytes into the tumorinterstitium.

[0589] By 24 h after coaguligand injection, the blood vessels were stilloccluded and extensive hemorrhage had spread throughout the tumor. Tumorcells had separated from one another, had pyknotic nuclei and wereundergoing cytolysis. By 72 h, advanced necrosis was evident throughoutthe tumor. Necrosis was clearly present in the intratumoral region ofthe tumor, where VCAM-1 expression on the vessels was not originallyprominent. The coaguligand binding was evidently effective to curtailblood flow in all tumor regions, resulting in widespread tumordestruction. Furthermore, it is likely that the initialcoaguhlgand-induced thrombin deposition results in increased inductionof the VCAM-1 target antigen on central vessels, thus amplifyingtargeting and tumor destruction.

[0590] The thrombotic action of anti-VCAM-1·tTF on tumor vessels wasantigen specific. None of the control reagents administered atequivalent quantities (tTF alone, anti-VCAM-1 antibody alone, tTF plusanti-VCAM-1 antibody or the control coaguligand of irrelevantspecificity) caused thrombosis (Table 3). TABLE 3Anti-VCAM-1·tTF-Mediated Thrombosis in L540 Tumor Bearing MiceThrombosed Vessels (%)^(b) Heart and Other Treatment^(a) L540 Tumor LungOrgans Saline 0-2 0 0 TTF 0-2 0 0 Anti-VCAM-1 Antibody 0-2 0 0Anti-VCAM-1 Antibody + tTF 0-2 0 0 Control IgG·tTF 0-2 0 0Anti-VCAM-1·tTF (<0.3 cm3)^(c)  0-10 0 0 Anti-VCAM-1·tTF (>0.3 cm³)40-70 0 0

[0591] 2. Lack of Thrombosis of Normal Blood Vessels

[0592] In addition to the thrombosis of tumor blood vessels, this studyalso shows that intravenous administration of the anti-VCAM-1·tTFcoaguligand does not induce thrombosis of blood vessels in normalorgans.

[0593] Despite expression of VCAM-1 on vessels in the heart and lung ofnormal or L540 tumor-bearing mice (Table 1), thrombosis did not occurafter anti-VCAM-1·tTF coaguligand administration. No signs ofthrombosis, tissue damage or altered morphology were seen in 25 miceinjected with 5 to 45 μg of coaguligand 4 or 24 h earlier. There was anormal histological appearance of the heart and lung from the same mousethat had major tumor thrombosis. All other major organs (brain, liver,kidney, spleen, pancreas, intestine, testis) also had unalteredmorphology.

[0594] Frozen sections of organs and tumors from coaguligand-treatedmice gave coincident staining patterns when developed with either theanti-TF antibody, 10HI0, or an anti-rat IgG antibody and confirmed thatthe coaguligand had localized to vessels in the heart, lung and tumor.The intensity of staining was equal to that seen when coaguligand wasapplied directly to the sections at high concentrations followed bydevelopment with anti-TF or anti-rat IgG, indicating that saturation ofbinding had been attained in vivo.

[0595] These studies show that binding of coaguligand to VCAM-1 onnormal vasculature in heart and lung is not sufficient to inducethrombosis, and that tumor vasculature provides additional factors tosupport coagulation.

EXAMPLE VII In Vivo Tumor Destruction by Anti-VCAM-1 Coaguligand

[0596] A. Methods

[0597] Male CB17 SCID mice were injected subcutaneously with 1×10⁷ L540cells as described above. When the tumors had reached a volume of0.4-0.6 cm³, the mice were injected intravenously with either 20 μg ofanti-VCAM-1·tTF, 16 μg anti-VCAM-1 antibody, 4 μg tTF, a mixture of 16μg of anti-VCAM-1 antibody and 4 μg of tTF, 20 μg control IgG·tTF orsaline. In some studies, the treatment was given 3 times, on days 0, 4and 8. A minimum of 8 animals were treated in each group.

[0598] Animals were monitored daily for tumor measurements and bodyweight. Mice were sacrificed when tumors had reached a diameter of 2cm³, or earlier if tumors showed signs of necrosis or ulceration. Tumorvolume was calculated according to the formula: π/6×D×d², where D is thelarger tumor diameter and d is the smaller diameter. Differences intumor growth rates were tested for statistical significance using anon-parametric test (Mann-Whitney rank sum test) that makes noassumptions about tumor size being normally distributed (Gibbons, 1976).

[0599] B. Results

[0600] The anti-tumor activity of anti-VCAM-1·tTF coaguligand wasdetermined in SCID mice bearing 0.3-0.4 cm³ L540 tumors. The drug wasadministered i.v. 3 times at intervals of 4 days. The pooled resultsfrom 3 separate studies are presented in FIG. 2 and Table 4. Mean tumorvolume of anti-VCAM-1·tTF treated mice was significantly reduced at 21days of treatment (P<0.00 1) in comparison to all other groups. Nine ofa total of 15 mice treated with the specific coaguligand showed morethan 50% reduction in tumor volume. This effect was specific sinceunconjugated tTF, control IgG coaguligand and mixture of freeanti-VCAM-1 antibody and tTF did not affect tumor growth. TABLE 4Inhibition of Tumor Growth by Anti-VCAM-1 · tTF Coaguligand Mean tumorvolume (mm³)^(b) Tumor Growth P versus Treatment^(a) N Day 0 Day 21Index^(c) saline^(d) Saline 14 331 ± 61 2190 ± 210 6.91 — TTF 13 341 ±22 2015 ± 205 5.90 NS Anti-VCAM-1 16 363 ± 24 1920 ± 272 5.28 NSAnti-VCAM- 13 349 ± 42 2069 ± 362 5.92 NS 1 + tTF Control IgG · 8 324 ±30 2324 ± 304 7.17 NS tTF Anti-VCAM- 15 365 ± 28 1280 ± 130 3.50 <0.0011 · tTF #The treatment was repeated on day 4 and 7 after firstinjection.

EXAMPLE VIII Phosphatidylserine Expression on Tumor Blood Vessels

[0601] A. Methods

[0602] 1. Antibodies

[0603] Anti-phosphatidylserine (anti-PS) and anti-cardiolipinantibodies, both mouse monoclonal IgM antibodies, were produced asdescribed by Rote (Rote et al., 1993). Details of the characterizationof the anti-PS and anti-cardiolipin antibodies were also reported byRote et al. (1993, incorporated herein by reference).

[0604] 2. Dctcection of PS Expression on Vascular Endothelium

[0605] L540 tumor-bearinig mice were injected i.v. with 20 μg of eitheranti-PS or anti-cardiolipin mouse IgM antibodies. After 10 min., micewere anesthetized and their blood circulations were perfused withheparinized saline. Tumors and normal tissues were removed andsnap-frozen. Serial sections of organs and tumors were stained witheither HRP-labeled anti-mouse IgM for detection of anti-PS antibody orwith anti-VCAM-1 antibody followed by HRP-labeled anti-rat Ig.

[0606] To preserve membrane phospholipids on frozen sections, thefollowing protocol was developed. Animals were perfused with DPBScontaining 2.5 mM Ca²⁺. Tissues were mounted on3-aminopropyltriethoxysilane-coated slides and were stained within 24 h.No organic solvents, formaldehyde or detergents were used for fixationor washing of the slides. Slides were re-hydrated by DPBS containing 2.5mM Ca²⁺ and 0.2% gelatin. The same solution was also used to washsections to remove the excess of reagents. Sections were incubated withHRP-labeled anti-mouse IgM for 3.5 h at room temperature to detectanti-PS IgM.

[0607] B. Results

[0608] To explain the lack of thrombotic effect of anti-VCAM-1·tTF onVCAM-1 positive vasculature in heart and lungs, the inventors developeda concept of differential PS localization between normal and tumor bloodvessels. Specifically, they hypothesized that endothelial cells innormal tissues segregate PS to the inner surface of the plasma membranephospholipid bilayer, where it is unable to participate in thromboticreactions; whereas endothelial cells in tumors translocate PS to theexternal surface of the plasma membrane, where it can support thecoagulation action of the coaguligand. PS expression on the cell surfaceallows coagulation because it enables the attachment of coagulationfactors to the membrane and coordinates the assembly of coagulationinitiation complexes (Ortel et at., 1992).

[0609] The inventors' model of PS translocation to the surface of tumorblood vessel endothelial cells, as developed herein, is surprising inthat PS expression does not occur after, and does not inevitablytrigger, cell death. PS expression at the tumor endothelial cell surfaceis thus sufficiently stable to allow PS to serve as a targetable entityfor therapeutic intervention.

[0610] To confirm the hypothesis that tumor blood vessel endotheliumexpresses PS on the luminal surface of the plasma membrane, theinventors used immunohistochemistry to determine the distribution ofaiiti-PS antibody after intravenous injection into L540 tumor bearingmice. Anti-PS antibody localized within 10 min. to the majority of tumorblood vessels, including vessels in the central region of the tumor thatcan lack VCAM-1. Vessels that were positive for VCAM-1 were alsopositive for PS. Thus, there is coincident expression of PS onVCAM-1-expressing vessels in tumors.

[0611] In the in vivo localization studies, none of the vessels innormal organs, including VCAM-1-positive vasculature of heart and lung,were stained, indicating that PS is absent from the external surface ofthe endothelial cells. In contrast, when sections of normal tissues andtumors were directly stained with anti-PS antibody in vitro, nodifferences were visible between normal and tumor, endothelial or othercell types, showing that PS is present within these cells but onlybecomes expressed on the surface of endothelial cells in tumors.

[0612] The specificity of PS detection was confirmed by two independentstudies. First, a mouse IgM monoclonal antibody directed against adifferent negatively charged lipid, cardiolipin, did not home to tumoror any organs in vivo. Second, pretreatment of frozen sections withacetone abolished staining with anti-PS antibody, presumably because itextracted the lipids together with the bound anti-PS antibody.

EXAMPLE IX Annexin V Blocks Coaguligand Activation of Factor X In Vitro

[0613] A. Methods

[0614] The ability of Annexin V to affect Factor Xa formation induced bycoaguligand was determined by a chromogenic assay described above inExample V. IL-1α-stimulated bEnd.3 cells were incubated withanti-VCAM-·tTF and permeabilized by saponin. Annexin V was added atconcentrations ranging from 0.1 to 10 μg/ml and cells were incubated for30 min. before addition of diluted Proplex T. The amount of Factor Xagenerated in the presence or absence of Annexin V was determined asdescribed in Example V. Each treatment was performed in duplicate andrepeated at least twice.

[0615] B. Results

[0616] The need for surface PS expression in coaguligand action isfurther indicated by the inventors' finding that annexin V, which bindsto PS with high affinity, blocks the ability of anti-VCAM -1·tTF boundto bEnd.3 cells to generate factor Xa in vitro.

[0617] Annexin V added to permeabilized cells preincubated withanti-VCAM-1·tTF inhibited the formation of factor Xa in a dose-dependentmanner (FIG. 3). In the absence of Annexin V, cell-bound coaguligandproduced 95 ng of factor Xa per 10,000 cells per 60 min. The addition ofincreasing amounts of Annexin V (in the tg per ml range) inhibitedfactor Xa production. At 10 μg per ml, Annexin V inhibited factor Xaproduction by 58% (FIG. 3). No further inhibition was observed byincreasing the concentration of Annexin V during the assay, indicatingthat annexin V saturated all available binding sites at 10 μg per ml.

EXAMPLE X Annexin V Blocks Coaguligand Activity In Vivo

[0618] A. Methods

[0619] The ability of Annexin V to inhibit coaguligand-inducedthrombosis in vivo was examined in L540 Hodgkin-bearing SCID mice.Tumors were grown in mice as described above in Example II. Two mice pergroup (tumor size 0.5 cm in diameter) were injected intravenously viathe tail vein with one of the following reagents: a) saline; b) 100 μgof Annexin V; c) 40 μg of anti-VCAM-1·tTF; d) 100 μg of Annexin Vfollowed 2 hours later by 40 μg of anti-VCAM-1·TF.

[0620] Four hours after the last injection mice were anesthetized andperfused with heparinized saline. Tumors were removed, fixed with 4%formalin, paraffin-embedded and stained with hematoxylene-eosin . Thenumber of thromboscd and non-thrombosed blood vessels were counted andthe percentage of thrombosis was calculated.

[0621] B. Results

[0622] Annexin V also blocks the activity of the anti-VCAM-1·tTFcoaguligand in vivo. Groups of tumor-bearing mice were treated with oneof the control or test reagents, as described in the methods. The micewere given (a) saline; (b) 100 μg of Annexin V; (c) 40 μg ofanti-VCAM-1·tTF coaguligand; or (d) 100 μg of Annexin V followed 2 hourslater by 40 μg of anti-VCAM-1·tTF coaguligand. Identical results wereobtained in both mice per group.

[0623] No spontaneous thrombosis, hemorrhages or necrosis were observedin tumors derived from saline-injected mice. Treatment with Annexin Valone did not alter tumor morphology.

[0624] In accordance with other data presented herein, 40 μg ofanti-VCAM-1·tTF coaguligand caused thrombosis in 70% of total tumorblood vessels. The majority of blood vessels were occluded with packederythrocytes and clots, and tumor cells were separated from one another.Both coaguligand-induced anti-tumor effects, i.e., intravascularthrombosis and changes in tumor cell morphology, were completelyabolished by pre-treating the mice with Annexin V.

[0625] These findings confirm that the anti-tumor effects ofcoaguligands are mediated through the blockage of tumor vasculature.These data also demonstrate that PS is essential for coaguligand-inducedthrombosis in vivo.

EXAMPLE XI Externalized Phosphatidylserine is a Global Marker of TumorBlood Vessels

[0626] A. Methods

[0627] PS exposure on tumor and normal vascular endothelium was examinedin three animal tumor models: L540 Hodgkin lymphoma, NCl-H358 non-smallcell lung carcinoma, and HT 29 colon adenocarcinoma (ATCC). To grow thetumors in vivo, 2×10⁶ cells were injected into the right flank of SCIDmice and allowed to reach 0.8-1.2 cm in diameter. Mice bearing largetumors (volume above 800 mm³) were injected intravenously via the tailvein with 20 μg of either anti-PS or anti-cardiolipin antibodies. Theanti-cardiolipin antibody served as a control for all studies since bothantibodies are directed against negatively charged lipids and belong tothe same class of immunoglobulins (mouse IgM).

[0628] One hour after injection, mice were anesthetized and their bloodcirculation was perfused with heparinized saline. Tumors and normalorgans were removed and snap-frozen. Frozen sections were stained withanti-mouse IgM-peroxidase conjugate (Jackson Immunoresearch Labs)followed by development with carbazole.

[0629] B. Results

[0630] The anti-PS antibodies specifically homed to the vasculature ofall three tumors (HT 29, L540 and NCl-H358) in vivo, as indicated bydetection of the mouse IgM. The average percentages of vessels stainedin the tumors were 80% for HT 29, 30% for L540 and 50% for NCl-H358.Vessels in all regions of the tumors were stained and there was stainingboth of small capillaries and larger vessels.

[0631] No vessel staining was observed with anti-PS antibodies in anynormal tissues. In the kidney, tubules were stained both with anti-PSand anti-CL, and this likely relates to the secretion of IgMs by thisorgan (Table 5). Anti-cardiolipin antibodies were not detected in anytumors or normal tissues, except kidney.

[0632] These findings indicate that only tumor endothelium exposes PS tothe outer site of the plasma membrane. TABLE 5 Vessel Localization ofAnti-PS and Anti-Cardiolipin Abs in Tumor-Bearing Mice* Tissue Anti-PS†Anti-Cardiolipin† L540 Cy tumor ++ − H358 tumor ++ − HT29 tumor +++ −Adrenal − − Brain Cerebellum − − Brain Cortex − − Heart − − Kidney −‡ −‡Large Intestine − − Liver − − Lung − − Pancreas − − Small Intestine − −Spleen − − Testes − −

[0633] To estimate the time at which tumor vasculature loses the abilityto segregate PS to the inner side of the membrane, the inventorsexamined anti-PS localization in L540 tumors ranging in volume from 140to 1,600 mm³. Mice were divided into 3 groups according to their tumorsize: 140-300, 350-800 and 800-1,600 mm³. Anti-PS Ab was not detected inthree mice bearing small L540 tumors (up to 300 mm³). Anti-PS Ablocalized in 3 animals of 5 in the group of intermediate size L540tumors and in all mice (4 out of 4) bearing large L540 tumors (Table 6).Percent of PS-positive blood vessels from total (identified by panendothelial marker Meca 32) was 10-20% in the L540 intermediate groupand 20-40% in the group of large L540 tumors (Table 6). TABLE 6 PSExternalization Detected in Mid and Large Sized Tumors Tumor %PS-Positive Vessels/ Size (mm³) No. Positive Tumors/Total* Total†350-800 3/5 10-20   850-1,600 4/4 20-40

EXAMPLE XII Anti-Tumor Effects of Unconjugated Anti-PhosphatidylserineAntibodies

[0634] A. Methods

[0635] The effects of anti-PS antibodies were examined in syngeneic andxenogeneic tumor models. For the syngeneic model, 1×10⁷ cells of murinecolorectal carcinoma Colo 26 (obtained from Dr. Ian Hart, ICRF, London)were injected subcutaneously into the right flank of Balb/c mice. In thexenogeneic model, a human Hodgkin's lymphoma L540 xenograft wasestablished by injecting IxI07 cells subcutaneously into the right flankof male CB17 SCID mice. Tumors were allowed to grow to a size of about0.6-0.9 cm³ before treatment.

[0636] Tumor-bearing mice (4 animals per group) were injected i.p. with20 μg of naked anti-PS antibody (lgM), control mouse IgM or saline.Treatment was repeated 3 times with a 48 hour interval. Animals weremonitored daily for tumor measurements and body weight. Tumor volume wascalculated as described in Example Vii. Mice were sacrificed when tumorshad reached 2 cm′, or earlier if tumors showed signs of necrosis orulceration.

[0637] B. Results

[0638] The growth of both syngeneic and xenogenieic tumors waseffectively inhibited by treatment with naked anti-PS antibodies (FIG.4A and FIG. 4B). Anti-PS antibodies caused tumor vascular injury,accompanied by thrombosis, and tumor necrosis. The presence of clots anddisintegration of tumor mass surrounding blocked blood vessels wasevident.

[0639] Quantitatively, the naked anti-PS antibody treatment inhibitedtumor growth by up to 60% of control tumor volume in mice bearing largeColo 26 (FIG. 4A) and L540 (FIG. 4B) tumors. No retardation of tumorgrowth was found in mice treated with saline or control IgM. No toxicitywas observed in mice treated with anti-PS antibodies, with normal organspreserving unaltered morphology, indistinguishable from untreated orsaline-treated mice.

[0640] Tumor regression started 24 hours after the first treatment andtumors continue to decline in size for the next 6 days. This wasobserved in both syngeneic and immunocompromised tumor models,indicating that the effect was mediated by immune status-independentmechanism(s). Moreover, the decline in tumor burden was associated withthe increase of alertness and generally healthy appearance of theanimals, compared to control mice bearing tumors larger than 1500 mm′.Tumor re-growth occurred 7-8 days after the first treatment.

[0641] The results obtained with anti-PS treatment of L540 tumors arefurther compelling for the following reasons. Notably, the tumornecrosis observed in L540 tumor treatment occurred despite the fact thatthe percentage of vessels that stained positive for PS in L540 tumorswas less than in HT 29 and NCl-H358 tumors. This implies that even morerapid necrosis would likely result when treating other tumor types.Furthermore, L540 tumors are generally chosen as an experimental modelbecause they provide clean histological sections and they are, in fact,known to be resistant to necrosis.

EXAMPLE XIII Anti-Tumor Effects of Annexin Conjugates

[0642] The surprising finding that aminophospholipids are stable markersof tumor vasculature also means that antibody-therapeutic agentconstructs can be used in cancer treatment. In addition to usingantibodies as targeting agents, the inventors reasoned that annexins,and other aminophospholipid-binding proteins, could also be used tospecifically deliver therapeutic agents to tumor vasculature. Thefollowing data shows the anti-tumor effects that result from the in vivoadministration of annexin-TF constructs.

[0643] A. Methods

[0644] An annexin V-tTF conjugate was prepared and administered to nu/numice with solid tumors. The tumors were formed from human HT29colorectal carcinoma cells that formed tumors of at least about 1.2 cm³.The annexin V-tTF coaguligand (10 μg) was administered intravenously andallowed to circulate for 24 hours. Saline-treated mice were separatelymaintained as control animals. After the one day treatment period, themice were sacrificed and exsanguinated and the tumors and major organswere harvested for analysis.

[0645] B. Results

[0646] The annexin V-tTF conjugate was found to induce specific tumorblood vessel coagulation in HT29 tumor bearing mice. Approximately 55%of the tumor blood vessels in the annexin V-tTF conjugate treatedanimals were thrombosed following a single injection. In contrast, therewas minimal evidence of thrombosis in the tumor vasculature of thecontrol animals.

EXAMPLE XIV Phosphatidylserine Translocation in the Tumor Environment

[0647] The discovery of PS as an in vivo surface marker unique to tumorvascular endothelial cells prompted the inventors to further investigatethe effect of a tumor environment on PS translocation and outer membraneexpression. The present example shows that exposing endothelial cells invitro to certain conditions that mimic those in a tumor duplicates theobserved PS surface expression in intact, viable cells.

[0648] A. Methods

[0649] Mouse bEnd.3 endothelial cells were seeded at an initial densityof 50,000 cells/well. Twenty-fours later cells were incubated withincreasing concentrations of H₂O₂ (from 10 μM to 500 μM) for 1 hour at37° C. or left untreated. At the end of the incubation, cells werewashed 3 times with PBS containing 0.2% gelatin and fixed with 0.25%glutaraldehyde. Identical wells were either stained with anti-PS IgM ortrypsinized and evaluated for viability by the Trypan Blue exclusiontest. For the anti-PS staining, after blocking with 2% gelatin for 10min., cells were incubated with 2 pig/ml of anti-PS antibody, followedby detection with anti-mouse IgM-HRP conjugate.

[0650] Wells seeded with mouse bEnd.3 endothelial cells were alsoincubated with different effectors and compared to control, untreatedwells after the same period of incubation at 37° C. The panel ofeffectors tested included TNF, LPS, bFGF, IL-1α, IL-1β and thrombin.After incubation, cells were washed and fixed and were again eitherstained with anti-PS IgM or evaluated for viability using the TrypanBlue exclusion test, as described above.

[0651] B. Results

[0652] 1. PS Induction by H₂O₂

[0653] Exposing endothelial cells to H₂O₂ at concentrations higher than100 μM caused PS translocation in 90% cells. However, this wasaccompanied by detachment of the cells from the substrate and cellviability decreasing to about 50-60%. The association of surface PSexpression with decreasing cell viability is understandable, although itis still interesting to note that ˜90% PS translocation is observed withonly a 50-60% decrease in cell viability.

[0654] Using concentrations of H₂O₂ lower than 100 μM resulted insignificant PS expression without any appreciable reduction in cellviability. For example, PS was detected at the cell surface of about 50%of cells in all H₂O₂ treated wells using H₂O₂ at concentrations as lowas 20 μM. It is important to note that, under these low H₂O₂concentrations, the cells remained firmly attached to the plastic and toeach other, showed no morphological changes and had no signs ofcytotoxicity. Detailed analyses revealed essentially 100% cell-cellcontact, retention of proper cell shape and an intact cytoskeleton.

[0655] The 50% PS surface expression induced by low levels of H₂O₂ wasthus observed in cell populations in which cell viability was identicalto the control, untreated cells (i e., 95%). The PS expressionassociated with high H₂O₂ concentrations was accompanied by cell damage,and the PS-positive cells exposed to over 100 μM H₂O₂ were detached,floating and had disrupted cytoskeletons.

[0656] The maintenance of cell viability in the presence of lowconcentrations H₂O₂ is consistent with data from other laboratories. Forexample, Schorer et al. (1985) showed that human umbilical veinendothelial cells (HUVEC) treated with 15 μM H₂O₂ averaged 90 to 95%viability (reported as 5% to 10% injury), whilst those exposed to 1500μM H₂O₂ were only 0%-50% viable (50% to 100% injured).

[0657] The use of H₂O₂ to mimic the tumor environment in vitro is alsoappropriate in that the tumor environment is rich in inflammatory cells,such as macrophages, PMNs and granulocytes, which produce H₂O₂ and otherreactive oxygen species. Although never before connected with stabletumor vascular markers, inflamimatory cells are known to mediateendothelial cell injury by mechanisms involving reactive oxygen speciesthat require the presence of H₂O₂ (Weiss et al., 1981; Yamada et al.,1981; Schorer et al., 1985). In fact, studies have shown thatstimulation of PMNs in vitro produces concentrations of H₂O₂ sufficientto cause sublethal endothelial cell injury without causing cell death(measured by chromium release assays) or cellular detachment; and thatthese H₂O₂ concentrations are attainable locally in vivo (Schorer etal., 1985).

[0658] The present in vitro translocation data correlates with theearlier results showing that anti-PS antibodies localize specifically totumor vascular endothelial cells in vivo, and do not bind to cells innormal tissues. The finding that in vivo-like concentrations of H₂O₂induce PS translocation to the endothelial cell surface withoutdisrupting cell integrity has important implications in addition tovalidating the original in vivo data and the inventors' therapeuticapproaches.

[0659] Human, bovine and murine endothelial cells are all known to bePS-negative under normal conditions. Any previously documented PSexpression has always been associated with cell damage and/or celldeath. This is simply not the case in the present studies, where normalviability is maintained. This shows that PS translocation in tumorvascular endothelium is mediated by biochemical mechanisms unconnectedto cell damage. This is believed to be the first demonstration of PSsurface expression in morphologically intact endothelial cells and thefirst indication that PS expression can be disconnected from theapoptosis pathway(s). Returning to the operability of the presentinvention, these observations again confirm that PS is a sustainable,rather than transient, marker of tumor blood vessels and a suitablecandidate for therapeutic intervention.

[0660] 2. PS Expression Does Not Correlate with Cell Activation

[0661] The relevance of this in vitro data to the tumor environment isalso strengthened by the fact that other, general cell activators arewithout effect on PS translocation in endothelial cells. For example,the inventors tested TNF in similarly controlled studies and found itunable to induce PS surface expression, despite the expected increasesin E-selectin and VCAM expression. Likewise, LPS, bFGF, IL-1α and IL-1βwere all without effect on PS expression in appropriately controlledstudies.

[0662] 3. PS Induction by Thrombin

[0663] In contrast to the lack of effects of other cell activators,thrombin was observed to increase PS expression, although not to thesame extent as H₂O₂. This data is also an integral part of thetumor-induction model of PS expression developed by the presentinventors (thrombin-induced PS surface expression in normal tissueswould also further coagulation as PS expression coordinates the assemblyof coagulation initiation complexes (Ortel et al., 1992)).

[0664] The tumor environment is known to be prothrombotic, such thattumor vasculature is predisposed to coagulation (U.S. Pat. No.5,877,289). As thrombin is a product of the coagulation cascade, it ispresent in tumor vasculature. In fact, the presence of thrombin inducesVCAM expression, contributing to the inventors' ability to exploit VCAMas a targetable marker of tumor vasculature (U.S. Pat. Nos. 5,855,866;5,877,289). The present data showing that thrombin also induces PSexpression is thus both relevant to targeting aminophospholipids withnaked antibodies and therapeutic conjugates, and further explains thebeneficial effects of the anti-VCAM coaguligand containing Tissue Factor(Example VII).

[0665] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1 5 1 2149 DNA Homo sapiens 1 cagctgactc aggcaggctc catgctgaacggtcacacag agaggaaaca ataaatctca 60 gctactatgc aataaatatc tcaagttttaacgaagaaaa acatcattgc agtgaaataa 120 aaaattttaa aattttagaa caaagctaacaaatggctag ttttctatga ttcttcttca 180 aacgctttct ttgaggggga aagagtcaaacaaacaagca gttttacctg aaataaagaa 240 ctagttttag aggtcagaag aaaggagcaagttttgcgag aggcacggaa ggagtgtgct 300 ggcagtacaa tgacagtttt cctttcctttgctttcctcg ctgccattct gactcacata 360 gggtgcagca atcagcgccg aagtccagaaaacagtggga gaagatataa ccggattcaa 420 catgggcaat gtgcctacac tttcattcttccagaacacg atggcaactg tcgtgagagt 480 acgacagacc agtacaacac aaacgctctgcagagagatg ctccacacgt ggaaccggat 540 ttctcttccc agaaacttca acatctggaacatgtgatgg aaaattatac tcagtggctg 600 caaaaacttg agaattacat tgtggaaaacatgaagtcgg agatggccca gatacagcag 660 aatgcagttc agaaccacac ggctaccatgctggagatag gaaccagcct cctctctcag 720 actgcagagc agaccagaaa gctgacagatgttgagaccc aggtactaaa tcaaacttct 780 cgacttgaga tacagctgct ggagaattcattatccacct acaagctaga gaagcaactt 840 cttcaacaga caaatgaaat cttgaagatccatgaaaaaa acagtttatt agaacataaa 900 atcttagaaa tggaaggaaa acacaaggaagagttggaca ccttaaagga agagaaagag 960 aaccttcaag gcttggttac tcgtcaaacatatataatcc aggagctgga aaagcaatta 1020 aacagagcta ccaccaacaa cagtgtccttcagaagcagc aactggagct gatggacaca 1080 gtccacaacc ttgtcaatct ttgcactaaagaaggtgttt tactaaaggg aggaaaaaga 1140 gaggaagaga aaccatttag agactgtgcagatgtatatc aagctggttt taataaaagt 1200 ggaatctaca ctatttatat taataatatgccagaaccca aaaaggtgtt ttgcaatatg 1260 gatgtcaatg ggggaggttg gactgtaatacaacatcgtg aagatggaag tctagatttc 1320 caaagaggct ggaaggaata taaaatgggttttggaaatc cctccggtga atattggctg 1380 gggaatgagt ttatttttgc cattaccagtcagaggcagt acatgctaag aattgagtta 1440 atggactggg aagggaaccg agcctattcacagtatgaca gattccacat aggaaatgaa 1500 aagcaaaact ataggttgta tttaaaaggtcacactggga cagcaggaaa acagagcagc 1560 ctgatcttac acggtgctga tttcagcactaaagatgctg ataatgacaa ctgtatgtgc 1620 aaatgtgccc tcatgttaac aggaggatggtggtttgatg cttgtggccc ctccaatcta 1680 aatggaatgt tctatactgc gggacaaaaccatggaaaac tgaatgggat aaagtggcac 1740 tacttcaaag ggcccagtta ctccttacgttccacaacta tgatgattcg acctttagat 1800 ttttgaaagc gcaatgtcag aagcgattatgaaagcaaca aagaaatccg gagaagctgc 1860 caggtgagaa actgtttgaa aacttcagaagcaaacaata ttgtctccct tccagcaata 1920 agtggtagtt atgtgaagtc accaaggttcttgaccgtga atctggagcc gtttgagttc 1980 acaagagtct ctacttgggg tgacagtgctcacgtggctc gactatagaa aactccactg 2040 actgtcgggc tttaaaaagg gaagaaactgctgagcttgc tgtgcttcaa actactactg 2100 gaccttattt tggaactatg gtagccagatgataaatatg gttaatttc 2149 2 498 PRT Homo sapiens 2 Met Thr Val Phe LeuSer Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys SerAsn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg IleGln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp GlyAsn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu GlnArg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys LeuGln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln LysLeu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105 110 Ala GlnIle Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 GluIle Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150155 160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln165 170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys AsnSer 180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His LysGlu Glu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln GlyLeu Val Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys GlnLeu Asn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val Leu Gln Lys GlnGln Leu Glu Leu Met Asp 245 250 255 Thr Val His Asn Leu Val Asn Leu CysThr Lys Glu Gly Val Leu Leu 260 265 270 Lys Gly Gly Lys Arg Glu Glu GluLys Pro Phe Arg Asp Cys Ala Asp 275 280 285 Val Tyr Gln Ala Gly Phe AsnLys Ser Gly Ile Tyr Thr Ile Tyr Ile 290 295 300 Asn Asn Met Pro Glu ProLys Lys Val Phe Cys Asn Met Asp Val Asn 305 310 315 320 Gly Gly Gly TrpThr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp 325 330 335 Phe Gln ArgGly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser 340 345 350 Gly GluTyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln 355 360 365 ArgGln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg 370 375 380Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn 385 390395 400 Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser405 410 415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala AspAsn 420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly GlyTrp Trp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met PheTyr Thr Ala 450 455 460 Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys TrpHis Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu Arg Ser Thr ThrMet Met Ile Arg Pro Leu 485 490 495 Asp Phe 3 2269 DNA Homo sapiens 3tgggttggtg tttatctcct cccagccttg agggagggaa caacactgta ggatctgggg 60agagaggaac aaaggaccgt gaaagctgct ctgtaaaagc tgacacagcc ctcccaagtg 120agcaggactg ttcttcccac tgcaatctga cagtttactg catgcctgga gagaacacag 180cagtaaaaac caggtttgct actggaaaaa gaggaaagag aagactttca ttgacggacc 240cagccatggc agcgtagcag ccctgcgttt cagacggcag cagctcggga ctctggacgt 300gtgtttgccc tcaagtttgc taagctgctg gtttattact gaagaaagaa tgtggcagat 360tgttttcttt actctgagct gtgatcttgt cttggccgca gcctataaca actttcggaa 420gagcatggac agcataggaa agaagcaata tcaggtccag catgggtcct gcagctacac 480tttcctcctg ccagagatgg acaactgccg ctcttcctcc agcccctacg tgtccaatgc 540tgtgcagagg gacgcgccgc tcgaatacga tgactcggtg cagaggctgc aagtgctgga 600gaacatcatg gaaaacaaca ctcagtggct aatgaagctt gagaattata tccaggacaa 660catgaagaaa gaaatggtag agatacagca gaatgcagta cagaaccaga cggctgtgat 720gatagaaata gggacaaacc tgttgaacca aacagctgag caaacgcgga agttaactga 780tgtggaagcc caagtattaa atcagaccac gagacttgaa cttcagctct tggaacactc 840cctctcgaca aacaaattgg aaaaacagat tttggaccag accagtgaaa taaacaaatt 900gcaagataag aacagtttcc tagaaaagaa ggtgctagct atggaagaca agcacatcat 960ccaactacag tcaataaaag aagagaaaga tcagctacag gtgttagtat ccaagcaaaa 1020ttccatcatt gaagaactag aaaaaaaaat agtgactgcc acggtgaata attcagttct 1080tcaaaagcag caacatgatc tcatggagac agttaataac ttactgacta tgatgtccac 1140atcaaactca gctaaggacc ccactgttgc taaagaagaa caaatcagct tcagagactg 1200tgctgaagta ttcaaatcag gacacaccac aaatggcatc tacacgttaa cattccctaa 1260ttctacagaa gagatcaagg cctactgtga catggaagct ggaggaggcg ggtggacaat 1320tattcagcga cgtgaggatg gcagcgttga ttttcagagg acttggaaag aatataaagt 1380gggatttggt aacccttcag gagaatattg gctgggaaat gagtttgttt cgcaactgac 1440taatcagcaa cgctatgtgc ttaaaataca ccttaaagac tgggaaggga atgaggctta 1500ctcattgtat gaacatttct atctctcaag tgaagaactc aattatagga ttcaccttaa 1560aggacttaca gggacagccg gcaaaataag cagcatcagc caaccaggaa atgattttag 1620cacaaaggat ggagacaacg acaaatgtat ttgcaaatgt tcacaaatgc taacaggagg 1680ctggtggttt gatgcatgtg gtccttccaa cttgaacgga atgtactatc cacagaggca 1740gaacacaaat aagttcaacg gcattaaatg gtactactgg aaaggctcag gctattcgct 1800caaggccaca accatgatga tccgaccagc agatttctaa acatcccagt ccacctgagg 1860aactgtctcg aactattttc aaagacttaa gcccagtgca ctgaaagtca cggctgcgca 1920ctgtgtcctc ttccaccaca gagggcgtgt gctcggtgct gacgggaccc acatgctcca 1980gattagagcc tgtaaacttt atcacttaaa cttgcatcac ttaacggacc aaagcaagac 2040cctaaacatc cataattgtg attagacaga acacctatgc aaagatgaac ccgaggctga 2100gaatcagact gacagtttac agacgctgct gtcacaacca agaatgttat gtgcaagttt 2160atcagtaaat aactggaaaa cagaacactt atgttataca atacagatca tcttggaact 2220gcattcttct gagcactgtt tatacactgt gtaaataccc atatgtcct 2269 4 496 PRTHomo sapiens 4 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu ValLeu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser IleGly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr PheLeu Leu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr ValSer Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser ValGln Arg Leu 65 70 75 80 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr GlnTrp Leu Met Lys 85 90 95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys GluMet Val Glu Ile 100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala ValMet Ile Glu Ile Gly 115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu GlnThr Arg Lys Leu Thr Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln ThrThr Arg Leu Glu Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser ThrAsn Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile AsnLys Leu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu AlaMet Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu GluLys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile IleGlu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235 240 AsnSer Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265270 Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275280 285 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn290 295 300 Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly GlyGly 305 310 315 320 Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser ValAsp Phe Gln 325 330 335 Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly AsnPro Ser Gly Glu 340 345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln LeuThr Asn Gln Gln Arg 355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp TrpGlu Gly Asn Glu Ala Tyr 370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu SerSer Glu Glu Leu Asn Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu ThrGly Thr Ala Gly Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn AspPhe Ser Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys CysSer Gln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly ProSer Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460 Asn Thr AsnLys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480 GlyTyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 4955 495 PRT Homo sapiens 5 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys AspLeu Val Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met AspSer Ile Gly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser TyrThr Phe Leu Leu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser ProTyr Val Ser Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp PheSer Ser Gln Lys Leu 65 70 75 80 Gln His Leu Glu His Val Met Glu Asn TyrThr Gln Trp Leu Gln Lys 85 90 95 Leu Glu Asn Tyr Ile Val Glu Asn Met LysSer Glu Met Ala Gln Ile 100 105 110 Gln Gln Asn Ala Val Gln Asn His ThrAla Thr Met Leu Glu Ile Gly 115 120 125 Thr Ser Leu Leu Ser Gln Thr AlaGlu Gln Thr Arg Lys Leu Thr Asp 130 135 140 Val Glu Thr Gln Val Leu AsnGln Thr Ser Arg Leu Glu Ile Gln Leu 145 150 155 160 Leu Glu Asn Ser LeuSer Thr Tyr Lys Leu Glu Lys Gln Leu Leu Gln 165 170 175 Gln Thr Asn GluIle Leu Lys Ile His Glu Lys Asn Ser Leu Leu Glu 180 185 190 His Lys IleLeu Glu Met Glu Gly Lys His Lys Glu Glu Leu Asp Thr 195 200 205 Leu LysGlu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr Arg Gln Thr 210 215 220 TyrIle Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala Thr Thr Asn 225 230 235240 Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp Thr Val His 245250 255 Asn Leu Val Asn Leu Ser Thr Lys Glu Gly Val Leu Leu Lys Gly Gly260 265 270 Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp Val TyrGln 275 280 285 Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile AsnAsn Met 290 295 300 Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val AsnGly Gly Gly 305 310 315 320 Trp Thr Val Ile Gln His Arg Glu Asp Gly SerLeu Asp Phe Gln Arg 325 330 335 Gly Trp Lys Glu Tyr Lys Met Gly Phe GlyAsn Pro Ser Gly Glu Tyr 340 345 350 Trp Leu Gly Asn Glu Phe Ile Phe AlaIle Thr Ser Gln Arg Gln Tyr 355 360 365 Met Leu Arg Ile Glu Leu Met AspTrp Glu Gly Asn Arg Ala Tyr Ser 370 375 380 Gln Tyr Asp Arg Phe His IleGly Asn Glu Lys Gln Asn Tyr Arg Leu 385 390 395 400 Tyr Leu Lys Gly HisThr Gly Thr Ala Gly Lys Gln Ser Ser Leu Ile 405 410 415 Leu His Gly AlaAsp Phe Ser Thr Lys Asp Ala Asp Asn Asp Asn Cys 420 425 430 Met Cys LysCys Ala Leu Met Leu Thr Gly Gly Trp Trp Phe Asp Ala 435 440 445 Cys GlyPro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala Gly Gln Asn 450 455 460 HisGly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys Gly Pro Ser 465 470 475480 Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu Asp Phe 485 490495

what is claimed is:
 1. A method for killing tumor vascular endothelialcells, comprising administering to an animal having a vascularized tumora biologically effective amount of at least a first antibody, orantigen-binding region thereof, that binds to an aminophospholipid onthe luminal surface of tumor vascular endothelial cells.
 2. A method forinducing coagulation in tumor vasculature, comprising administering toan animal having a vascularized tumor a vessel-occluding amount of atleast a first antibody, or antigen-binding region thereof, that binds toan aminophospholipid on the luminal surface of tumor vasculature.
 3. Amethod for destroying tumor vasculature, comprising administering to ananimal having a vascularized tumor a tumor-destructive amount of atleast a first antibody, or antigen-binding region thereof, that binds toan aminophospholipid on the luminal surface of tumor vasculature.
 4. Amethod for treating an anima having a vascularized tumor, comprisingadministering to said animal a theraputically effective amount of atleast a first pharmaceutical composition comprising at least a firstantibody or antigen-binding fragment thereof, that binds to anaminophospholipid on the luminal surface of blod vessels of thevascularized tumor.
 5. The method of claim 4, wherein saidpharmaceitical composition comprises at least a first antibody, orantigen-binding fragment thereof, that binds to phosphatidylethanolamineon the luminal surface of blood vessels of the vascularized tumor. 6.The method of claim 4, wherein said pharmaceutical composition comprisesat least a first antibody, or antigen-binding fragment thereof, thatbinds to phosphatidylserine on the luminal surface of blood vessels ofthe vascularized tumor.
 7. The method of claim 4, wherein saidpharmaceutical composition comprises at least a first IgG or IgManti-aminophospholipid antibody.
 8. The method of claim 4, wherein saidpharmaceutical composition comprises at least a first scFv, Fv, Fab′,Fab or F(ab′)₂ antigen-binding fragment of an anti-aminophospholipidantibody.
 9. The method of claim 4, wherein said pharmaceuticalcomposition comprises at least a first human, humanized or part-humanchimeric anti-aminophospholipid antibody or antigen-binding fragmentthereof.
 10. The method of claim 4, wherein said phamaceuticalcomposition comprises at least a first anti-aminophospholipid monoclonalantibody or antigen-binding fragment thereof.
 11. The method of claim10, wherein said pharmaceutical composition comprises at least a firstanti-aminophospholipid monoclonal antibody, or antigen-binding fragmentthereof, that is prepared by a preparative process comprising: (a)preparing an anti-aminophospholipid antibody-producing cell; and (b)obtaining an anti-aminophospholipid monoclonal antibody from saidantibody-producing cell.
 12. The method of claim 11, wherein saidanti-aminophospholipid antibody-producing cell is obtained from a humanpatient having a disease associated with the production ofanti-aminophospholipid antibodies.
 13. The method of claim 11, whereinsaid anti-aminophospholipid antibody-producing cell is obtained bystimulating a mixed population of human peripheral blood lymphocyteswith an immunogenically effective amount of an aminophospholipid sample.14. The method of claim 11, wherein said anti-aminophospholipidantibody-producing cell Is obtained by immunizing an animal with animmunogenically effective amount of an aminophospholipid sample.
 15. Themethod of claim 14, wherein said anti-aminophospholipidantibody-producing cell is obtained by immunizing a transgenic mousethat comprises a human antibody library with an immunogenicallyeffective amount of an aminophosphoipid sample.
 16. The method of claim11, wherein said preparative process comprises: (a) fusing saidanti-aminophospholipid antibody-producing cell with an immortal cell toprepare a hybridoma that produces an anti-aminophospholipid monoclonalantibody; and (b) obtaining an anti-arninopliospholipid monoclonalantibody from said hybridoma.
 17. The method of claim 11, wherein saidpreparative process comprises: (a) immunizing an animal with animmunogenically effective amount of an aminophospholipid sample; (b)preparing a collection of antibody-producing hybridomas from theimmunized animal; (c) selecting from the collection a hybridoma thatproduces an anti-aminophospholipid antibody; and (d) culturing theselected hybridoma to provide the anti-aminophospholipid monoclonalantibody.
 18. The method of claim 17, wherein an antigen-binding regionof the anti-aminophospholipid monoclonal antibody is operativelyattached to a human antibody framework or constant region.
 19. Themethod of claim 17, wherein the immunized animal is a transgenic mousethat comprises a human antibody library and wherein theanti-aminophospholipid monoclonal antbody is a human monoclonalantibody.
 20. The method of claim 11, wherein said preparative processcomprises: (a) obtaining anti-aminophospholipid antibody-encodinignuLcleic acids from said anti-aminophospholipid antibody-producing cell;and (b) expressing said nucleic acids to obtain a recombinantanti-aminophospholipid monoclonal antibody.
 21. The method of claim 11,wherein said preparative process comprises: (a) immunizing an animalwith an immunogenically effective amount of an aminophospholipid sample;(b) preparing a combinatorial immunoglobulin phagemid library expressingRNA isolated from the spleen of the immunized animal; (c) selecting fromthe phagemid library a clone that expresses an anti-aminophospholipidantibody; and (d) expressing an anti-aminophospholipidantibody-encoding, nucleic acid from said selected clone to provide arecombinant anti-aminophospholipid monoclonal antibody.
 22. The methodof claim 21, wherein the immunized animal is a transgenic mouse thatcomprises a human antibody library and wherein the recombinantanti-aminophospholipid monoclonal antibody is a recombinant humanmonoclonal antibody.
 23. The method of claim 4, wherein saidpharmaceutical composition comprises a dimer, trimer or mlliimer of ananti-aminophospholipid antibody or antigen binding fragments thereof.24. The method of claim 4, wherein at least a second antibody that bindsto an aminophosphiolipid, or an antigen-binding fragment thereof, isadministered to said animal.
 25. The method of claim 4, wherein saidpharmaceutical composition is administered to said animal viaintravenous administration.
 26. The method of claim 4, wherein an imageof the vasculature of said vascularized tumor is first obtained byadministering to said animal a diagnostically effective amount of adetectably-labeled antibody, or antigen-binding fragment thereof, thatbinds to and identifies an aminophospholipid on the luminal surface ofblood vessels of the vascularized tumor.
 27. The method of claim 4,further comprising subjecting said animal to surgery or radiotherapy.28. The method of claim 4, further comprising simultaneously orsequentially administering to said animal a therapeutically effectiveamount of at least a second anti-cancer agent.
 29. The method of claim28, wherein said at least a second anti-cancer agent is achemotherapeutic, radiotherapeutic, anti-angiogenic orapoptosis-inducing agent.
 30. The method of claim 28, wherein said atleast a second anti-cancer agent is an antibody-therapeutic agentconstruct comprising a targeting antibody, or antigen-binding fragmentthereof, that binds to a surface-expressed, surface-accessible orsurface-localized component of a tumor cell, tumor stroma or tumorvasculature; said targeting antibody or fragment thereof operativelylinked to a therapeutic agent.
 31. The method of claim 30, wherein saidtargeting antibody, or antigen-binding fragment thereof, binds to a cellsurface antigen of a tumor cell.
 32. The method of claim 30, whereinsaid targeting antibody, or antigen-binding fragment thereof, binds to acomponent of tumor stroma.
 33. The method of claim 30, wherein saidtargeting antibody, or antigen-binding fragment thereof, binds to asurface-expressed, surface-accessible, surface-localized,cytokine-inducible or cagulant-inducible component of intratumoral bloodvessels of a vascularized tumor.
 34. The method of claim 33, whereinsaid targeting antibody, or antigen-binding fragment thereof, binds to asurface-expressed component of intratumoral vasculature selected fromthe group consisting of an aminophospholipid, endoglin, a TGFβ receptor,E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGFNVPF receptor, anFGF receptor, a TIE, α_(v)β₃ integrin, pleiotropin, endosialin and anMHC Class II protein.
 35. The method of claim 33, wherein said targetingantibody, or antigen-binding fragment thereof, binds to asurface-localized component of intratumoral vasculature selected fromthe 30 group consisting of VEGFNVPF, FGF, TGFβ, a ligand that binds to aTIE, a tumor-associated fibronectin isoform, scatter factor/hepatocytegrowth factor (HGF), platelet factor 4 (PF4), PDGF and TIMP.
 36. Themethod of claim 30, wherein said targeting antibody, or antigen-bindingfragment thereof, is operatively linked to a cytotoxic agent.
 37. Themethod of claim 36, wherein said targeting antibody, or antigen-bindingfragment thereof, is operatively linked to a plant-, fungus- orbacteria-derived toxin.
 38. The method of claim 37, wherein saidtargeting antibody, or antigen-binding fragment thereof, is operativelylinked to deglycosytated ricin A chain.
 39. The method of claim 30,wherein said targeting antibody, or antigen-binding fragment thereof, isoperatively linked to a coagulation factor or to an antibody, orantigen-binding fragment thereof, that binds to a coagulation factor.40. The method of claim 39, wherein said targeting antibody, orantigen-binding fragment thereof, is operatively linked to TissueFactor, truncated Tissue Factor or a derivative thereof, or to anantibody, or antigen-binding fragment thereof, that binds to TissueFactor, truncated Tissue Factor or a derivative thereof.
 41. The methodof claim 4, wherein said animal is a human patient.
 42. A method fortreating cancer. comprising administering to an animal having avascularized tumor a therapeutically etfective amount of at least afirst pharmaceutical composition comprising at least a first nakedantibody, or antigen-binding fragment thereof, that binds to anaminophospholipid on the luminal surface of intratumoral blood vesselsof the vascularized tumor.
 43. A method for treating cancer, comprisingadministering to an animal having a vascularized tumor at least a firstpharmaceutical composition comprising an amount of at least a firstunconjugated antibody effective to kill at least a portion of the tumorvascular endothelial cells; wherein said first unconjugated antibody isan unconjugated antibody, or antigen-binding fragment thereof, thatbinds to an aminophospholipid expressed on the luminal surface of tumorvascular endothelial cells.
 44. A method for treating cancer, comprisingadministering to an animal having a vascularized tumor at least a firstpharmaceutical composition comprising an amount of at least a firstunconjugated antibody effective to occlude or destroy tumor bloodvessels, as opposed to normal blood vessels; wherein said firstunconjugated antibody is an unconjugated antibody, or antigen-bindingfragment thereof, that binds to an aminophospholipid expressed on theluminal surface of tumor vascular endothelial cells.
 45. A method fortreating cancer, comprising administering to an animal having avascularized tumor at least a first pharmaceutical compositioncomprising an amount of at least a first unconjugated antibody effectiveto induce tumor necrosis; wherein said first unconjugated antibody is anunconjugated antibody, or antigen-binding fragment thereof, that bindsto an aminophospholipid expressed on the luminal surface of bloodvessels of the vascularized tumor.
 46. A method for treating cancer,comprising: (a) forming an image of a vascularized tumor byadministering to an animal having a vascularized tumor a diagnosticallyeffective amount of a detectably-labeled antibody, or antigen-bindingfragment thereof, that binds to an aminophospholipid on the luminalsurface of blood vessels of the vascularized tumor, thereby forming adetectable image of the tumor vasculature; and (b) subsequentlyadministering to said animal a therapeutically effective amount of atleast a first antibody, or antigen-binding fragment thereof, that bindsto an aminophospholipid on the tumor blood vessel luminal surface andthereby destroys the tumor vasculature.
 47. A method for treatingcancer, comprising simultaneously or sequentially administering to ananimal having a vascularized tumor a therapeutically effectivecombination of an unconjugated antibody, or antigen-binding fragmentthereof, that binds to an aminophospholipid on the luminal surface ofblood vessels of the vascularized tumor and at least a secondanti-cancer agent.
 48. The method of claim 47, wherein said at least asecond anti-cancer agent is a chemotherapeutic, radiotherapeutic,anti-angiogenic or apoptosis-inducing agent or an antibody-therapeuticagent construct comprising a therapeutic agent operatively attached toan antibody, or antigen-binding fragment thereof, that binds to asurface-expressed, surface-accessible, surface-localized,cytokine-inducible or coagulant-inducible component of tumor vasculatureor tumor stroma.